Sulfurylase-luciferase fusion proteins and thermostable sulfurylase

ABSTRACT

The present invention relates to the field of DNA recombinant technology. More specifically, this invention relates to fusion proteins comprising an ATP generating polypeptide joined to a polypeptide that converts ATP into a detectable entity. Accordingly, this invention focuses on sulfurylase-luciferase fusion proteins. This invention also relates to pharmaceutical compositions containing the fusion proteins and methods for using them.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 60/335,949 filedOct. 30, 2001; U.S. Pat. No. 60/349,076 filed Jan. 16, 2002; and U.S.Ser. No. 10/122,706 filed Apr. 11, 2002. This application is acontinuation-in-part application of U.S. application Ser. No.10/122,706. The contents of these applications are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to fusion proteins that are useful asreporter proteins, in particular to fusion proteins of ATP sulfurylaseand luciferase which are utilized to achieve an efficient conversion ofpyrophosphate (PPi) to light. This invention also relates to a novelthermostable sulfurylase which can be used in the detection of inorganicpyrophosphate, particularly in the sequencing of nucleic acid.

BACKGROUND OF THE INVENTION

ATP sulfurylase has been identified as being involved in sulfurmetabolism. It catalyzes the initial reaction in the metabolism ofinorganic sulfate (SO₄ ⁻²); see e.g., Robbins and Lipmann, 1958. J.Biol. Chem. 233: 686-690; Hawes and Nicholas, 1973. Biochem. J. 133:541-550). In this reaction SO₄ ⁻² is activated to adenosine5′-phosphosulfate (APS). ATP sulfurylase is also commonly used inpyrophosphate sequencing methods. In order to convert pyrophosphate(PPi) generated from the addition of dNMP to a growing DNA chain tolight, PPi must first be converted to ATP by ATP sulfurylase.

ATP produced by an ATP sulfurylase can also be hydrolyzed usingenzymatic reactions to generate light. Light-emitting chemical reactions(i.e., chemiluminescence) and biological reactions (i.e.,bioluminescence) are widely used in analytical biochemistry forsensitive measurements of various metabolites. In bioluminescentreactions, the chemical reaction that leads to the emission of light isenzyme-catalyzed. For example, the luciferin-luciferase system allowsfor specific assay of ATP. Thus, both ATP generating enzymes, such asATP sulfurylase, and light emitting enzymes, such as luciferase, couldbe useful in a number of different assays for the detection and/orconcentration of specific substances in fluids and gases. Since highphysical and chemical stability is sometimes required for enzymesinvolved in sequencing reactions, a thermostable enzyme is desirable.

Because the product of the sulfurylase reaction is consumed byluciferase, proximity between these two enzymes by covalently linkingthe two enzymes in the form of a fusion protein would provide for a moreefficient use of the substrate. Substrate channeling is a phenomenon inwhich substrates are efficiently delivered from enzyme to enzyme withoutequilibration with other pools of the same substrates. In effect, thiscreates local pools of metabolites at high concentrations relative tothose found in other areas of the cell. Therefore, a fusion of an ATPgenerating polypeptide and an ATP converting peptide could benefit fromthe phenomenon of substrate channeling and would reduce production costsand increase the number of enzymatic reactions that occur during a giventime period.

All patents and publications cited throughout the specification arehereby incorporated by reference into this specification in theirentirety in order to more fully describe the state of the art to whichthis invention pertains.

SUMMARY OF THE INVENTION

The invention provides a fusion protein comprising an ATP generatingpolypeptide bound to a polypeptide which converts ATP into an entitywhich is detectable. In one aspect, the invention provides a fusionprotein comprising a sulfurylase polypeptide bound to a luciferasepolypeptide. This invention provides a nucleic acid that comprises anopen reading frame that encodes a novel thermostable sulfurylasepolypeptide. In a further aspect, the invention provides for a fusionprotein comprising a thermostable sulfurylase joined to at least oneaffinity tag.

In another aspect, the invention provides a recombinant polynucleotidethat comprises a coding sequence for a fusion protein having asulfurylase poylpeptide sequence joined to a luciferase polypeptidesequence. In a further aspect, the invention provides an expressionvector for expressing a fusion protein. The expression vector comprisesa coding sequence for a fusion protein having: (i) a regulatorysequence, (ii) a first polypeptide sequence of an ATP generatingpolypeptide and (iii) a second polypeptide sequence that converts ATP toan entity which is detectable. In an additional embodiment, the fusionprotein comprises a sulfurylase polypeptide and a luciferasepolypeptide. In another aspect, the invention provides a transformedhost cell which comprises the expression vector. In an additionalaspect, the invention provides a fusion protein bound to a mobilesupport. The invention also includes a kit comprising asulfurylase-luciferase fusion protein expression vector.

The invention also includes a method for determining the nucleic acidsequence in a template nucleic acid polymer, comprising: (a) introducingthe template nucleic acid polymer into a polymerization environment inwhich the nucleic acid polymer will act as a template polymer for thesynthesis of a complementary nucleic acid polymer when nucleotides areadded; (b) successively providing to the polymerization environment aseries of feedstocks, each feedstock comprising a nucleotide selectedfrom among the nucleotides from which the complementary nucleic acidpolymer will be formed, such that if the nucleotide in the feedstock iscomplementary to the next nucleotide in the template polymer to besequenced said nucleotide will be incorporated into the complementarypolymer and inorganic pyrophosphate will be released; (c) separatelyrecovering each of the feedstocks from the polymerization environment;and (d) measuring the amount of PPi with an ATP generatingpolypeptide-ATP converting polypeptide fusion protein in each of therecovered feedstocks to determine the identity of each nucleotide in thecomplementary polymer and thus the sequence of the template polymer. Inone embodiment, the amount of inorganic pyrophosphate is measured by thesteps of: (a) adding adenosine-5′-phosphosulfate to the feedstock; (b)combining the recovered feedstock containing adenosine-5′-phosphosulfatewith an ATP generating polypeptide-ATP converting polypeptide fusionprotein such that any inorganic pyrophosphate in the recovered feedstockand the adenosine-5′-phosphosulfate will react to the form ATP andsulfate; (c) combining the ATP and sulfate-containing feedstock withluciferin in the presence of oxygen such that the ATP is consumed toproduced AMP, inorganic pyrophosphate, carbon dioxide and light; and (d)measuring the amount of light produced.

In another aspect, the invention includes a method wherein eachfeedstock comprises adenosine-5′-phosphosulfate and luciferin inaddition to the selected nucleotide base, and the amount of inorganicpyrophosphate is determined by reacting the inorganic pyrophosphatefeedstock with an ATP generating polypeptide-ATP converting polypeptidefusion protein thereby producing light in an amount proportional to theamount of inorganic pyrophosphate, and measuring the amount of lightproduced.

In another aspect, the invention provides a method for sequencing anucleic acid, the method comprising: (a) providing one or more nucleicacid anchor primers; (b) providing a plurality of single-strandedcircular nucleic acid templates disposed within a plurality of cavitieson a planar surface, each cavity forming an analyte reaction chamber,wherein the reaction chambers have a center to center spacing of between5 to 200 μm; (c) annealing an effective amount of the nucleic acidanchor primer to at least one of the single-stranded circular templatesto yield a primed anchor primer-circular template complex; (d) combiningthe primed anchor primer-circular template complex with a polymerase toform an extended anchor primer covalently linked to multiple copies of anucleic acid complementary to the circular nucleic acid template; (e)annealing an effective amount of a sequencing primer to one or morecopies of said covalently linked complementary nucleic acid; (f)extending the sequencing primer with a polymerase and a predeterminednucleotide triphosphate to yield a sequencing product and, if thepredetermined nucleotide triphosphate is incorporated onto the 3′ end ofsaid sequencing primer, a sequencing reaction byproduct; and (g)identifying the sequencing reaction byproduct with the use of a ATPgenerating polypeptide-ATP converting polypeptide fusion protein,thereby determining the sequence of the nucleic acid.

In one aspect, the invention provides a method for sequencing a nucleicacid, the method comprising: (a) providing at least one nucleic acidanchor primer; (b) providing a plurality of single-stranded circularnucleic acid templates in an array having at least 400,000 discretereaction sites; (c) annealing a first amount of the nucleic acid anchorprimer to at least one of the single-stranded circular templates toyield a primed anchor primer-circular template complex; (d) combiningthe primed anchor primer-circular template complex with a polymerase toform an extended anchor primer covalently linked to multiple copies of anucleic acid complementary to the circular nucleic acid template; (e)annealing a second amount of a sequencing primer to one or more copiesof the covalently linked complementary nucleic acid; (f) extending thesequencing primer with a polymerase and a predetermined nucleotidetriphosphate to yield a sequencing product and, when the predeterminednucleotide triphosphate is incorporated onto the 3′ end of thesequencing primer, to yield a sequencing reaction byproduct; and (g)identifying the sequencing reaction byproduct with the use of a ATPgenerating polypeptide-ATP converting polypeptide fusion protein,thereby determining the sequence of the nucleic acid at each reactionsite that contains a nucleic acid template.

In another aspect, the invention includes a method of determining thebase sequence of a plurality of nucleotides on an array, the methodcomprising the steps of: (a) providing a plurality of sample DNAs, eachdisposed within a plurality of cavities on a planar surface, each cavityforming an analyte reaction chamber, wherein the reaction chambers havea center to center spacing of between 5 to 200 μm, (b) adding anactivated nucleotide 5′-triphosphate precursor of one known nitrogenousbase to a reaction mixture in each reaction chamber, each reactionmixture comprising a template-directed nucleotide polymerase and asingle-stranded polynucleotide template hybridized to a complementaryoligonucleotide primer strand at least one nucleotide residue shorterthan the templates to form at least one unpaired nucleotide residue ineach template at the 3′-end of the primer strand, under reactionconditions which allow incorporation of the activated nucleoside5′-triphosphate precursor onto the 3′-end of the primer strands,provided the nitrogenous base of the activated nucleoside5′-triphosphate precursor is complementary to the nitrogenous base ofthe unpaired nucleotide residue of the templates; (c) determiningwhether or not the nucleoside 5′-triphosphate precursor was incorporatedinto the primer strands through detection of a sequencing byproduct witha ATP generating polypeptide-ATP converting polypeptide fusion protein,thus indicating that the unpaired nucleotide residue of the template hasa nitrogenous base composition that is complementary to that of theincorporated nucleoside 5′-triphosphate precursor; and (d) sequentiallyrepeating steps (b) and (c), wherein each sequential repetition addsand, detects the incorporation of one type of activated nucleoside5′-triphosphate precursor of known nitrogenous base composition; and (e)determining the base sequence of the unpaired nucleotide residues of thetemplate in each reaction chamber from the sequence of incorporation ofsaid nucleoside precursors.

In one aspect, the invention includes a method for determining thenucleic acid sequence in a template nucleic acid polymer, comprising:(a) introducing a plurality of template nucleic acid polymers into aplurality of cavities on a planar surface, each cavity forming ananalyte reaction chamber, wherein the reaction chambers have a center tocenter spacing of between 5 to 200 μm, each reaction chamber having apolymerization environment in which the nucleic acid polymer will act asa template polymer for the synthesis of a complementary nucleic acidpolymer when nucleotides are added; (b) successively providing to thepolymerization environment a series of feedstocks, each feedstockcomprising a nucleotide selected from among the nucleotides from whichthe complementary nucleic acid polymer will be formed, such that if thenucleotide in the feedstock is complementary to the next nucleotide inthe template polymer to be sequenced said nucleotide will beincorporated into the complementary polymer and inorganic pyrophosphatewill be released; (c) detecting the formation of inorganic pyrophosphatewith an ATP generating polypeptide-ATP converting polypeptide fusionprotein to determine the identify of each nucleotide in thecomplementary polymer and thus the sequence of the template polymer.

In one aspect, the invention provides a method of identifying the basein a target position in a DNA sequence of sample DNA including the stepscomprising: (a) disposing sample DNA within a plurality of cavities on aplanar surface, each cavity forming an analyte reaction chamber, whereinthe reaction chambers have a center to center spacing of between 5 to200 μm, said DNA being rendered single stranded either before or afterbeing disposed in the reaction chambers, (b) providing an extensionprimer which hybridizes to said immobilized single-stranded DNA at aposition immediately adjacent to said target position; (c) subjectingsaid immobilized single-stranded DNA to a polymerase reaction in thepresence of a predetermined nucleotide triphosphate, wherein if thepredetermined nucleotide triphosphate is incorporated onto the 3′ end ofsaid sequencing primer then a sequencing reaction byproduct is formed;and

(d) identifying the sequencing reaction byproduct with a ATP generatingpolypeptide-ATP converting polypeptide fusion protein, therebydetermining the nucleotide complementary to the base at said targetposition.

The invention also includes a method of identifying a base at a targetposition in a sample DNA sequence comprising: (a) providing sample DNAdisposed within a plurality of cavities on a planar surface, each cavityforming an analyte reaction chamber, wherein the reaction chambers havea center to center spacing of between 5 to 200 μm, said DNA beingrendered single stranded either before or after being disposed in thereaction chambers; (b) providing an extension primer which hybridizes tothe sample DNA immediately adjacent to the target position; (c)subjecting the sample DNA sequence and the extension primer to apolymerase reaction in the presence of a nucleotide triphosphate wherebythe nucleotide triphosphate will only become incorporated and releasepyrophosphate (PPi) if it is complementary to the base in the targetposition, said nucleotide triphosphate being added either to separatealiquots of sample-primer mixture or successively to the samesample-primer mixture; (d) detecting the release of PPi with an ATPgenerating polypeptide-ATP converting polypeptide fusion protein toindicate which nucleotide is incorporated.

In one aspect, the invention provides a method of identifying a base ata target position in a single-stranded sample DNA sequence, the methodcomprising: (a) providing an extension primer which hybridizes to sampleDNA immediately adjacent to the target position, said sample DNAdisposed within a plurality of cavities on a planar surface, each cavityforming an analyte reaction chamber, wherein the reaction chambers havea center to center spacing of between 5 to 200 um, said DNA beingrendered single stranded either before or after being disposed in thereaction chambers; (b) subjecting the sample DNA and extension primer toa polymerase reaction in the presence of a predetermined deoxynucleotideor dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotidewill only become incorporated and release pyrophosphate (PPi) if it iscomplementary to the base in the target position, said predetermineddeoxynucleotides or dideoxynucleotides being added either to separatealiquots of sample-primer mixture or successively to the samesample-primer mixture, (c) detecting any release of PPi with an ATPgenerating polypeptide-ATP converting polypeptide fusion protein toindicate which deoxynucleotide or dideoxynucleotide isincorporated;characterized in that, the PPi-detection enzyme(s) areincluded in the polymerase reaction step and in that in place of deoxy-or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is usedwhich is capable of acting as a substrate for a polymerase but incapableof acting as a substrate for a said PPi—detection enzyme.

In another aspect, the invention includes a method of determining thebase sequence of a plurality of nucleotides on an array, the methodcomprising: (a) providing a plurality of sample DNAs, each disposedwithin a plurality of cavities on a planar surface, each cavity formingan analyte reaction chamber, wherein the reaction chambers have a centerto center spacing of between 5 to 200 μm, (b) converting PPi into lightwith an ATP generating polypeptide-ATP converting polypeptide fusionprotein; (c) detecting the light level emitted from a plurality ofreaction sites on respective portions of an optically sensitive device;(d) converting the light impinging upon each of said portions of saidoptically sensitive device into an electrical signal which isdistinguishable from the signals from all of said other regions; (e)determining a light intensity for each of said discrete regions from thecorresponding electrical signal; (f) recording the variations of saidelectrical signals with time.

In one aspect, the invention provides a method for sequencing a nucleicacid, the method comprising:(a) providing one or more nucleic acidanchor primers; (b) providing a plurality of single-stranded circularnucleic acid templates disposed within a plurality of cavities on aplanar surface, each cavity forming an analyte reaction chamber, whereinthe reaction chambers have a center to center spacing of between 5 to200 μm;(c) converting PPi into a detectable entity with the use of anATP generating polypeptide-ATP converting polypeptide fusion protein;(d) detecting the light level emitted from a plurality of reaction siteson respective portions of an optically sensitive device; (e) convertingthe light impinging upon each of said portions of said opticallysensitive device into an electrical signal which is distinguishable fromthe signals from all of said other regions; (f) determining a lightintensity for each of said discrete regions from the correspondingelectrical signal; (g) recording the variations of said electricalsignals with time.

In another aspect, the invention includes a method for sequencing anucleic acid, the method comprising: (a) providing at least one nucleicacid anchor primer; (b) providing a plurality of single-strandedcircular nucleic acid templates in an array having at least 400,000discrete reaction sites; (c) converting PPi into a detectable entitywith an ATP generating polypeptide-ATP converting polypeptide fusionprotein; (d) detecting the light level emitted from a plurality ofreaction sites on respective portions of an optically sensitive device;(e) converting the light impinging upon each of said portions of saidoptically sensitive device into an electrical signal which isdistinguishable from the signals from all of said other regions; (f)determining a light intensity for each of said discrete regions from thecorresponding electrical signal; (g) recording the variations of saidelectrical signals with time.

In another aspect, the invention includes an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) a mature form of an amino acid sequence of SEQ ID NO: 2; (b) avariant of a mature form of an amino acid sequence of SEQ ID NO: 2; anamino acid sequence of SEQ ID NO: 2; (c) a variant of an amino acidsequence of SEQ ID NO: 2, wherein one or more amino acid residues insaid variant differs from the amino acid sequence of said mature form,provided that said variant differs in no more than 5% of amino acidresidues from said amino acid sequence; (d) and at least oneconservative amino acid substitution to the amino acid sequences in (a),(b), (c) or (d). The invention also includes an antibody that bindsimmunospecifically to the polypeptide of (a), (b), (c) or (d).

In another aspect, the invention includes an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) a mature form of an amino acid sequence of SEQ ID NO: 2; (b) avariant of a mature form of an amino acid sequence of SEQ ID NO: 2,wherein one or more amino acid residues in said variant differs from theamino acid sequence of said mature form, provided that said variantdiffers in no more than 5% of the amino acid residues from the aminoacid sequence of said mature form; (c) an amino acid sequence of SEQ IDNO: 2; (d) a variant of an amino acid sequence of SEQ ID NO: 2, whereinone or more amino acid residues in said variant differs from the aminoacid sequence of said mature form, provided that said variant differs inno more than 15% of amino acid residues from said amino acid sequence; anucleic acid fragment encoding at least a portion of a polypeptidecomprising an amino acid sequence of SEQ ID NO: 2, or a variant of saidpolypeptide, wherein one or more amino acid residues in said variantdiffers from the amino acid sequence of said mature form, provided thatsaid variant differs in no more than 5% of amino acid residues from saidamino acid sequence; (e) and a nucleic acid molecule comprising thecomplement of (a), (b), (c), (d) or (e).

In a further aspect, the invention provides a nucleic acid moleculewherein the nucleic acid molecule comprises nucleotide sequence selectedfrom the group consisting of: (a) a first nucleotide sequence comprisinga coding sequence differing by one or more nucleotide sequences from acoding sequence encoding said amino acid sequence, provided that no morethan 20% of the nucleotides in the coding sequence in said firstnucleotide sequence differ from said coding sequence; an isolated secondpolynucleotide that is a complement of the first polynucleotide; (b) anda nucleic acid fragment of (a) or (b). The invention also includes avector comprising the nucleic acid molecule of (a) or (b). In anotheraspect, the invention includes a cell comprising the vector.

In a further aspect, the invention includes a method for determining thenucleic acid sequence in a template nucleic acid polymer, comprising:(a) introducing the template nucleic acid polymer into a polymerizationenvironment in which the nucleic acid polymer will act as a templatepolymer for the synthesis of a complementary nucleic acid polymer whennucleotides are added; (b) successively providing to the polymerizationenvironment a series of feedstocks, each feedstock comprising anucleotide selected from among the nucleotides from which thecomplementary nucleic acid polymer will be formed, such that if thenucleotide in the feedstock is complementary to the next nucleotide inthe template polymer to be sequenced said nucleotide will beincorporated into the complementary polymer and inorganic pyrophosphatewill be released; (c) separately recovering each of the feedstocks fromthe polymerization environment; and (d) measuring the amount of PPi withan ATP sulfurylase and a luciferase in each of the recovered feedstocksto determine the identity of each nucleotide in the complementarypolymer and thus the sequence of the template polymer.

In another aspect, the invention provides a method for sequencing anucleic acid, the method comprising: (a) providing one or more nucleicacid anchor primers; (b) providing a plurality of single-strandedcircular nucleic acid templates disposed within a plurality of cavitiesin an array on a planar surface, each cavity forming an analyte reactionchamber, wherein the reaction chambers have a center to center spacingof between 5 to 200 μm and at least 400,000 discrete sites; (c)annealing an effective amount of the nucleic acid anchor primer to atleast one of the single-stranded circular templates to yield a primedanchor primer-circular template complex; (d) combining the primed anchorprimer-circular template complex with a polymerase to form an extendedanchor primer covalently linked to multiple copies of a nucleic acidcomplementary to the circular nucleic acid template; (e) annealing aneffective amount of a sequencing primer to one or more copies of saidcovalently linked complementary nucleic acid; (f) extending thesequencing primer with a polymerase and a predetermined nucleotidetriphosphate to yield a sequencing product and, if the predeterminednucleotide triphosphate is incorporated onto the 3′ end of saidsequencing primer, a sequencing reaction byproduct; and (g) identifyingthe sequencing reaction byproduct with the use of an ATP sulfurylase anda luciferase, thereby determining the sequence of the nucleic acid.

In another aspect, the invention provides a method for sequencing anucleic acid, the method comprising: (a) providing at least one nucleicacid anchor primer; (b) providing a plurality of single-strandedcircular nucleic acid templates in an array having at least 400,000discrete reaction sites; (c) annealing a first amount of the nucleicacid anchor primer to at least one of the single-stranded circulartemplates to yield a primed anchor primer-circular template complex; (d)combining the primed anchor primer-circular template complex with apolymerase to form an extended anchor primer covalently linked tomultiple copies of a nucleic acid complementary to the circular nucleicacid template; (e) annealing a second amount of a sequencing primer toone or more copies of the covalently linked complementary nucleic acid;(f) extending the sequencing primer with a polymerase and apredetermined nucleotide triphosphate to yield a sequencing product and,when the predetermined nucleotide triphosphate is incorporated onto the3′ end of the sequencing primer, to yield a sequencing reactionbyproduct; and (g) identifying the sequencing reaction byproduct withthe use of a thermostable sulfurylase and a luciferase, therebydetermining the sequence of the nucleic acid at each reaction site thatcontains a nucleic acid template.

In a further aspect, the invention includes a method of determining thebase sequence of a plurality of nucleotides on an array, the methodcomprising: (a) providing a plurality of sample DNAs, each disposedwithin a plurality of cavities on a planar surface, each cavity formingan analyte reaction chamber, wherein the reaction chambers have a centerto center spacing of between 5 to 200 μm, (b) adding an activatednucleotide 5′-triphosphate precursor of one known nitrogenous base to areaction mixture in each reaction chamber, each reaction mixturecomprising a template-directed nucleotide polymerase and asingle-stranded polynucleotide template hybridized to a complementaryoligonucleotide primer strand at least one nucleotide residue shorterthan the templates to form at least one unpaired nucleotide residue ineach template at the 3′-end of the primer strand, under reactionconditions which allow incorporation of the activated nucleoside5′-triphosphate precursor onto the 3′-end of the primer strands,provided the nitrogenous base of the activated nucleoside5′-triphosphate precursor is complementary to the nitrogenous base ofthe unpaired nucleotide residue of the templates; (c) detecting whetheror not the nucleoside 5′-triphosphate precursor was incorporated intothe primer strands through detection of a sequencing byproduct with athermostable sulfurylase and luciferase, thus indicating that theunpaired nucleotide residue of the template has a nitrogenous basecomposition that is complementary to that of the incorporated nucleoside5′-triphosphate precursor; and (d) sequentially repeating steps (b) and(c), wherein each sequential repetition adds and, detects theincorporation of one type of activated nucleoside 5′-triphosphateprecursor of known nitrogenous base composition; and (e) determining thebase sequence of the unpaired nucleotide residues of the template ineach reaction chamber from the sequence of incorporation of saidnucleoside precursors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is one embodiment for a cloning strategy for obtaining theluciferase-sulfurylase sequence.

FIGS. 2A and 2B show the preparative agarose gel of luciferase andsulfurylase as well as sulfurylase-luciferase fusion genes.

FIG. 3 shows the results of experiments to determine the activity of theluciferase-sulfurylase fusion protein on NTA-agarose and MPG-SA solidsupports.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a fusion protein containing an ATP generatingpolypeptide bound to a polypeptide which converts ATP into an entitywhich is detectable. As used herein, the term “fusion protein” refers toa chimeric protein containing an exogenous protein fragment joined toanother exogenous protein fragment. The fusion protein could include anaffinity tag to allow attachment of the protein to a solid support or toallow for purification of the recombinant fusion protein from the hostcell or culture supernatant, or both.

In a preferred embodiment, the ATP generating polypeptide and ATPconverting polypeptide are from a eukaryote or a prokaryote. Theeukaryote could be an animal, plant, fungus or yeast. In someembodiments, the animal is a mammal, rodent, insect, worm, mollusk,reptile, bird and amphibian. Plant sources of the polypeptides includebut are not limited to Arabidopsis thaliana, Brassica napus, Alliumsativum, Amaranthus caudatus, Hevea brasiliensis, Hordeum vulgare,Lycopersicon esculentum, Nicotiana tabacum, Oryza sativum, Pisumsativum, Populus trichocarpa, Solanum tuberosum, Secale cereale,Sambucus nigra, Ulmus americana or Triticum aestivum. Examples of fungiinclude but are not limited to Penicillum chrysogenum, Stachybotryschartarum, Aspergillus fumigatus, Podospora anserina and Trichodermareesei. Examples of sources of yeast include but are not limited toSaccharomyces cerevisiae, Candida tropicalis, Candida lypolitica,Candida utilis, Kluyveromyces lactis, Schizosaccharomyces pombe,Yarrowia lipolytica, Candida spp., Pichia spp. and Hansenula spp.

The prokaryote source could be bacteria or archaea. In some embodiments,the bacteria E. coli, B. subtilis, Streptococcus gordonii, flavobacteriaor green sulfur bacteria. In other embodiments, the archaea isSufolobus, Thermococcus, Methanobacterium, Halococcus, Halobacterium orMethanococcus jannaschii.

The ATP generating polypeptide can be a ATP sulfurylase, hydrolase or anATP synthase. In a preferred embodiment, the ATP generating polypeptideis ATP sulfurylase. In one embodiment, the ATP sulfurylase is athermostable sulfurylase cloned from Bacillus stearothermophilus (Bst)and comprising the nucleotide sequence of SEQ ID NO: 1. This putativegene was cloned using genomic DNA acquired from ATCC (Cat. No. 12980D).The gene is shown to code for a functional ATP sulfurylase that can beexpressed as a fusion protein with an affinity tag. The disclosed Bstsulfurylase nucleic acid (SEQ ID NO: 1) includes the 1247 nucleotidesequence. An open reading frame (ORF) for the mature protein wasidentified beginning with an ATG codon at nucleotides 1-3 and endingwith a TAA codon at nucleotides 1159-1161. The start and stop codons ofthe open reading frame are highlighted in bold type. The putativeuntranslated regions are underlined and found upstream of the initiationcodon and downstream from the termination codon.

Bst Thermostable Sulfurylase Nucleotide Sequence GTTATGAACATGAGTTTGAGCATTCCGCATGGCGGCACATTGATCAACCGTTGGAATCCG 60 (SEQ ID NO:1)GATTACCCAATCGATGAAGCAACGAAAACGATCGAGCTGTCCAAAGCCGAACTAAGCGAC 120CTTGAGCTGATCGGCACAGGCGCCTACAGCCCGCTCACCGGGTTTTTAACGAAAGCCGAT 180TACGATGCGGTCGTAGAAACGATGCGCCTCGCTGATGGCACTGTCTGGAGCATTCCGATC 240ACGCTGGCGGTGACGGAAGAAAAAGCGAGTGAACTCACTGTCGGCGACAAAGCGAAACTC 300GTTTATGGCGGCGACGTCTACGGCGTCATTGAAATCGCCGATATTTACCGCCCGGATAAA 360ACGAAAGAAGCCAAGCTCGTCTATAAAACCGATGAACTCGCTCACCCGGGCGTGCGCAAG 420CTGTTTGAAAAACCAGATGTGTACGTCGGCGGAGCGGTTACGCTCGTCAAACGGACCGAC 480AAAGGCCAGTTTGCTCCGTTTTATTTCGATCCGGCCGAAACGCGGAAACGATTTGCCGAA 540CTCGGCTGGAATACCGTCGTCGGCTTCCAAACACGCAACCCGGTTCACCGCGCCCATGAA 600TACATTCAAAAATGCGCGCTTGAAATCGTGGACGGCTTGTTTTTAAACCCGCTCGTCGGC 660GAAACGAAAGCGGACGATATTCCGGCCGACATCCGGATGGAAAGCTATCAAGTGCTGCTG 720GAAAACTATTATCCGAAAGACCGCGTTTTCTTGGGCGTCTTCCAAGCTGCGATGCGCTAT 780GCCGGTCCGCGCGAAGCGATTTTCCATGCCATGGTGCGGAAAAACTTCGGCTGCACGCAC 840TTCATCGTCGGCCGCGACCATGCGGGCGTCGGCAACTATTACGGCACGTATGATGCGCAA 900AAAATCTTCTCGAACTTTACAGCCGAAGAGCTTGGCATTACACCGCTCTTTTTCGAACAC 960AGCTTTTATTGCACGAAATGCGAAGGCATGGCATCGACGAAAACATGCCCGCACGACGCA 1020CAATATCACGTTGTCCTTTCTGGCACGAAAGTCCGTGAAATGTTGCGTAACGGCCAAGTG 1080CCGCCGAGCACATTCAGCCGTCCGGAAGTGGCCGCCGTTTTGATCAAAGGGCTGCAAGAA 1140CGCGAAACGGTCACCCCGTCGACACGCTAA AGGAGGAGCGAGATGAGCACGAATATCGTT 1200TGGCATCATACATCGGTGACAAAAGAAGATCGCCGCCAACGCAACGG 1247

The Bst sulfurylase polypeptide (SEQ ID NO:2) is 386 amino acid residuesin length and is presented using the three letter amino acid code.

Bst Sulfurylase Amino Acid Sequence Met Ser Leu Ser Ile Pro His Gly GlyThr Leu Ile Asn Arg Trp Asn (SEQ ID NO:2)1               5                    10                  15      Pro AspTyr Pro Ile Asp Glu Ala Thr Lys Thr Ile Glu Leu Ser Lys                20                  25                  30      Ala GluLeu Ser Asp Leu Glu Leu Ile Gly Thr Gly Ala Tyr Ser Pro            35                  40                  45          Leu ThrGly Phe Leu Thr Lys Ala Asp Tyr Asp Ala Val Val Glu Thr        50                  55                  60              Met ArgLeu Ala Asp Gly Thr Val Trp Ser Ile Pro Ile Thr Leu Ala    65                  70                  75                  Val ThrGlu Glu Lys Ala Ser Glu Leu Thr Val Gly Asp Lys Ala Lys80                  85                  90                  95  Leu ValTyr Gly Gly Asp Val Tyr Gly Val Ile Glu Ile Ala Asp Ile                100                 105                 110     Tyr ArgPro Asp Lys Thr Lys Glu Ala Lys Leu Val Tyr Lys Thr Asp            115                 120                 125         Glu LeuAla His Pro Gly Val Arg Lys Leu Phe Glu Lys Pro Asp Val        130                 135                 140             Tyr ValGly Gly Ala Val Thr Leu Val Lys Arg Thr Asp Lys Gly Gln    145                 150                 155                 Phe AlaPro Phe Tyr Phe Asp Pro Ala Glu Thr Arg Lys Arg Phe Ala160                 165                 170                 175 Glu LeuGly Trp Asn Thr Val Val Gly Phe Gln Thr Arg Asn Pro Val                180                 185                 190     His ArgAla His Glu Tyr Ile Gln Lys Cys Ala Leu Glu Ile Val Asp            195                 200                 205         Gly LeuPhe Leu Asn Pro Leu Val Gly Glu Thr Lys Ala Asp Asp Ile        210                 215                 220             Pro AlaAsp Ile Arg Met Glu Ser Tyr Gln Val Leu Leu Glu Asn Tyr    225                 230                 235                 Tyr ProLys Asp Arg Val Phe Leu Gly Val Phe Gln Ala Ala Met Arg240                 245                 250                 255 Tyr AlaGly Pro Arg Glu Ala Ile Phe His Ala Met Val Arg Lys Asn                260                 265                 270     Phe GlyCys Thr His Phe Ile Val Gly Arg Asp His Ala Gly Val Gly            275                 280                 285         Asn TyrTyr Gly Thr Tyr Asp Ala Gln Lys Ile Phe Ser Asn Phe Thr        290                 295                 300             Ala GluGlu Leu Gly Ile Thr Pro Leu Phe Phe Glu His Ser Phe Tyr    305                 310                 315                 Cys ThrLys Cys Glu Gly Met Ala Ser Thr Lys Tbr Cys Pro His Asp320                 325                 330                 335 Ala GlnTyr His Val Val Leu Ser Gly Thr Lys Val Arg Glu Met Leu                340                 345                 350     Arg AsnGly Gln Val Pro Pro Ser Thr Phe Ser Arg Pro Glu Val Ala            355                 360                 365         Ala ValLeu Ile Lys Gly Leu Gln Glu Arg Glu Thr Val Thr Pro Ser        370                 375                 380             Thr Arg    385

In one embodiment, the thermostable sulfurylase is active attemperatures above ambient to at least 50° C. This property isbeneficial so that the sulfurylase will not be denatured at highertemperatures commonly utilized in polymerase chain reaction (PCR)reactions or sequencing reactions. In one embodiment, the ATPsulfurylase is from a thermophile. The thermostable sulfurylase can comefrom thermophilic bacteria, including but not limited to, Bacillusstearothermophilus, Thermus thermophilus, Bacillus caldolyticus,Bacillus subtilis, Bacillus thermoleovorans, Pyrococcus furiosus,Sulfolobus acidocaldarius, Rhodothermus obamensis, Aquifex aeolicus,Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum,Pyrococcus abyssi, Penicillium chrysogenum, Sulfolobus solfataricus andThermomonospora fusca.

The homology of twelve ATP sulfurylases can be shown graphically in theClustalW analysis in Table 1. The alignment is of ATP sulfurylases fromthe following species: Bacillus stearothermophilus (Bst), University ofOklahoma—Strain 10 (Univ of OK), Aquifex aeolicus (Aae), Pyrococcusfuriosus (Pfu), Sulfolobus solataricus (Sso), Pyrobaculum aerophilum(Pae), Archaeoglobus fulgidus (Afu), Penicillium chrysogenum (Pch),Aeropyrum pernix (Ape), Saccharomyces cerevisiae (Sce), andThermomonospora fusca (Tfu).

TABLE 1 ClustalW Analysis of ATP Sulfurylase Amino Acid Sequence

A thermostable sulfurylase polypeptide is encoded by the open readingframe (“ORF”) of a thermostable sulfurylase nucleic acid. An ORFcorresponds to a nucleotide sequence that could potentially betranslated into a polypeptide. A stretch of nucleic acids comprising anORF is uninterrupted by a stop codon. An ORF that represents the codingsequence for a full protein begins with an ATG “start” codon andterminates with one of the three “stop” codons, namely, TAA, TAG, orTGA. For the purposes of this invention, an ORF may be any part of acoding sequence, with or without a start codon, a stop codon, or both.For an ORF to be considered as a good candidate for coding for abonafide cellular protein, a minimum size requirement is often set,e.g., a stretch of DNA that would encode a protein of 50 amino acids ormore.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequences shown in SEQ ID NO:1 due to degeneracy ofthe genetic code and thus encode the same thermostable sulfurylaseproteins as that encoded by the nucleotide sequences shown in SEQ IDNO:1. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence shown in SEQ ID NO:2. In addition to the thermostablesulfurylase nucleotide sequence shown in SEQ ID NO:1 it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequences of the thermostablesulfurylase polypeptides may exist within a population (e.g., thebacterial population). Such genetic polymorphism in the thermostablesulfurylase genes may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules comprising an openreading frame encoding a thermostable sulfurylase protein. Such naturalallelic variations can typically result in 1-5% variance in thenucleotide sequence of the thermostable sulfurylase genes. Any and allsuch nucleotide variations and resulting amino acid polymorphisms in thethermostable sulfurylase polypeptides, which are the result of naturalallelic variation and that do not alter the functional activity of thethermostable sulfurylase polypeptides, are intended to be within thescope of the invention.

Moreover, nucleic acid molecules encoding thermostable sulfurylaseproteins from other species, and thus that have a nucleotide sequencethat differs from the sequence SEQ ID NO:1 are intended to be within thescope of the invention. Nucleic acid molecules corresponding to naturalallelic variants and homologues of the thermostable sulfurylase cDNAs ofthe invention can be isolated based on their homology to thethermostable sulfurylase nucleic acids disclosed herein using the humancDNAs, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions. The invention further includes the nucleic acid sequence ofSEQ ID NO: 1 and mature and variant forms thereof, wherein a firstnucleotide sequence comprising a coding sequence differing by one ormore nucleotide sequences from a coding sequence encoding said aminoacid sequence, provided that no more than 11% of the nucleotides in thecoding sequence differ from the coding sequence.

Another aspect of the invention pertains to nucleic acid moleculesencoding a thermostable sulfurylase protein that contains changes inamino acid residues that are not essential for activity. Suchthermostable sulfurylase proteins differ in amino acid sequence from SEQID NO:2 yet retain biological activity. In separate embodiments, theisolated nucleic acid molecule comprises a nucleotide sequence encodinga protein, wherein the protein comprises an amino acid sequence at leastabout 96%, 97%, 98% or 99% homologous to the amino acid sequence of SEQID NO:2. An isolated nucleic acid molecule encoding a thermostablesulfurylase protein homologous to the protein of SEQ ID NO:2 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of SEQ ID NO:1 such that oneor more amino acid substitutions, additions or deletions are introducedinto the encoded protein.

Mutations can be introduced into SEQ ID NO:2 by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted, non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined withinthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted non-essentialamino acid residue in the thermostable sulfurylase protein is replacedwith another amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a thermostable sulfurylase codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for thermostable sulfurylase biological activity toidentify mutants that retain activity. Following mutagenesis of SEQ IDNO:1, the encoded protein can be expressed by any recombinant technologyknown in the art and the activity of the protein can be determined.

The relatedness of amino acid families may also be determined based onside chain interactions. Substituted amino acids may be fully conserved“strong” residues or fully conserved “weak” residues. The “strong” groupof conserved amino acid residues may be any one of the following groups:STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the singleletter amino acid codes are grouped by those amino acids that may besubstituted for each other. Likewise, the “weak” group of conservedresidues may be any one of the following: CSA, ATV, SAG, STNK, STPA,SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within eachgroup represent the single letter amino acid code.

The thermostable sulfurylase nucleic acid of the invention includes thenucleic acid whose sequence is provided herein, or fragments thereof.The invention also includes mutant or variant nucleic acids any of whosebases may be changed from the corresponding base shown herein whilestill encoding a protein that maintains its sulfurylase-like activitiesand physiological functions, or a fragment of such a nucleic acid. Theinvention further includes nucleic acids whose sequences arecomplementary to those just described, including nucleic acid fragmentsthat are complementary to any of the nucleic acids just described. Theinvention additionally includes nucleic acids or nucleic acid fragments,or complements thereto, whose structures include chemical modifications.Such modifications include, by way of nonlimiting example, modifiedbases, and nucleic acids whose sugar phosphate backbones are modified orderivatized. These modifications are carried out at least in part toenhance the chemical stability of the modified nucleic acid, such thatthey may be used, for example, as antisense binding nucleic acids intherapeutic applications in a subject.

A thermostable sulfurylase nucleic acid can encode a mature thermostablesulfurylase polypeptide. As used herein, a “mature” form of apolypeptide or protein disclosed in the present invention is the productof a naturally occurring polypeptide or precursor form or proprotein.The naturally occurring polypeptide, precursor or proprotein includes,by way of nonlimiting example, the full-length gene product, encoded bythe corresponding gene. Alternatively, it may be defined as thepolypeptide, precursor or proprotein encoded by an ORF described herein.The product “mature” form arises, again by way of nonlimiting example,as a result of one or more naturally occurring processing steps as theymay take place within the cell, or host cell, in which the gene productarises. Examples of such processing steps leading to a “mature” form ofa polypeptide or protein include the cleavage of the N-terminalmethionine residue encoded by the initiation codon of an ORF, or theproteolytic cleavage of a signal peptide or leader sequence. Thus amature form arising from a precursor polypeptide or protein that hasresidues 1 to N, where residue 1 is the N-terminal methionine, wouldhave residues 2 through N remaining after removal of the N-terminalmethionine. Alternatively, a mature form arising from a precursorpolypeptide or protein having residues 1 to N, in which an N-terminalsignal sequence from residue 1 to residue M is cleaved, would have theresidues from residue M+1 to residue N remaining. Further as usedherein, a “mature” form of a polypeptide or protein may arise from astep of post-translational modification other than a proteolyticcleavage event. Such additional processes include, by way ofnon-limiting example, glycosylation, myristoylation or phosphorylation.In general, a mature polypeptide or protein may result from theoperation of only one of these processes, or a combination of any ofthem.

The term “isolated” nucleic acid molecule, as utilized herein, is one,which is separated from other nucleic acid molecules which are presentin the natural source of the nucleic acid. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′- and 3′-termini of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated thermostablesulfurylase nucleic acid molecules can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of thecell/tissue from which the nucleic acid is derived (e.g., brain, heart,liver, spleen, etc.). Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orof chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the invention, e.g., a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1 or a complement of thisaforementioned nucleotide sequence, can be isolated using standardmolecular biology techniques and the sequence information providedherein. Using all or a portion of the nucleic acid sequence of SEQ IDNO:1 as a hybridization probe, thermostable sulfurylase molecules can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook, et al., (eds.), Molecular Cloning: A LaboratoryManual. 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel, et al., (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to thermostable sulfurylasenucleotide sequences can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, van der Waals, hydrophobic interactions, and the like.A physical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Fragments provided herein are defined as sequences of at least 6(contiguous) nucleic acids or at least 4 (contiguous) amino acids, alength sufficient to allow for specific hybridization in the case ofnucleic acids or for specific recognition of an epitope in the case ofamino acids, respectively, and are at most some portion less than a fulllength sequence. Fragments may be derived from any contiguous portion ofa nucleic acid or amino acid sequence of choice. Derivatives are nucleicacid sequences or amino acid sequences formed from the native compoundseither directly or by modification or partial substitution. Analogs arenucleic acid sequences or amino acid sequences that have a structuresimilar to, but not identical to, the native compound but differs fromit in respect to certain components or side chains. Analogs may besynthetic or from a different evolutionary origin and may have a similaror opposite metabolic activity compared to wild type. Homologs arenucleic acid sequences or amino acid sequences of a particular gene thatare derived from different species.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 89% identity over a nucleic acid or amino acid sequence ofidentical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art, orwhose encoding nucleic acid is capable of hybridizing to the complementof a sequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below.

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of thermostable sulfurylase polypeptides. Isoforms can beexpressed in different tissues of the same organism as a result of, forexample, alternative splicing of RNA. Alternatively, isoforms can beencoded by different genes. In the invention, homologous nucleotidesequences include nucleotide sequences encoding for a thermostablesulfurylase polypeptide of species other than humans, including, but notlimited to: vertebrates, and thus can include, e.g., frog, mouse, rat,rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotidesequences also include, but are not limited to, naturally occurringallelic variations and mutations of the nucleotide sequences set forthherein. Homologous nucleic acid sequences include those nucleic acidsequences that encode conservative amino acid substitutions in SEQ IDNO:1, as well as a polypeptide possessing thermostable sulfurylasebiological activity. Various biological activities of the thermostablesulfurylase proteins are described below.

The thermostable sulfurylase proteins of the invention include thesulfurylase protein whose sequence is provided herein. The inventionalso includes mutant or variant proteins any of whose residues may bechanged from the corresponding residue shown herein while still encodinga protein that maintains its sulfurylase-like activities andphysiological functions, or a functional fragment thereof. The inventionfurther encompasses antibodies and antibody fragments, such as F_(ab) or(F_(ab))₂, that bind immunospecifically to any of the proteins of theinvention. This invention also includes a variant or a mature form ofthe amino acid sequence of SEQ ID NO:2, wherein one or more amino acidresidues in the variant differs in no more than 4% of the amino acicresidues from the amino acid sequence of the mature form.

Several assays have been developed for detection of the forward ATPsulfurylase reaction. The colorimetric molybdolysis assay is based onphosphate detection (see e.g., Wilson and Bandurski, 1958. J. Biol.Chem. 233: 975-981), whereas the continuous spectrophotometricmolybdolysis assay is based upon the detection of NADH oxidation (seee.g., Seubert, et al, 1983. Arch. Biochem. Biophys. 225: 679-691;Seubert, et al, 1985. Arch. Biochem. Biophys. 240: 509-523). The laterassay requires the presence of several detection enzymes.

Suitable enzymes for converting ATP into light include luciferases,e.g., insect luciferases. Luciferases produce light as an end-product ofcatalysis. The best known light-emitting enzyme is that of the firefly,Photinus pyralis (Coleoptera). The corresponding gene has been clonedand expressed in bacteria (see e.g., de Wet, et al., 1985. Proc. Nat.Acad. Sci. USA 80: 7870-7873) and plants (see e.g., Ow, et al., 1986.Science 234: 856-859), as well as in insect (see e.g., Jha, et al.,1990. FEBS Lett. 274: 24-26) and mammalian cells (see e.g., de Wet, etal, 1987. Mol. Cell. Biol. 7: 725-7373; Keller, et al., 1987. Proc.Natl. Acad. Sci. USA 82: 3264-3268). In addition, a number of luciferasegenes from the Jamaican click beetle, Pyroplorus plagiophihalamus(Coleoptera), have recently been cloned and partially characterized (seee.g., Wood, et al., 1989. J. Biolumin. Chemilumin. 4: 289-301; Wood, etal., 1989. Science 244: 700-702). Distinct luciferases can sometimesproduce light of different wavelengths, which may enable simultaneousmonitoring of light emissions at different wavelengths. Accordingly,these aforementioned characteristics are unique, and add new dimensionswith respect to the utilization of current reporter systems.

Firefly luciferase catalyzes bioluminescence in the presence ofluciferin, adenosine 5′-triphosphate (ATP), magnesium ions, and oxygen,resulting in a quantum yield of 0.88 (see e.g., McElroy and Selinger,1960. Arch. Biochem. Biophys. 88: 136-145). The firefly luciferasebioluminescent reaction can be utilized as an assay for the detection ofATP with a detection limit of approximately 1×10⁻¹³ M (see e.g., Leach,1981. J. Appl. Biochem. 3: 473-517). In addition, the overall degree ofsensitivity and convenience of the luciferase-mediated detection systemshave created considerable interest in the development of fireflyluciferase-based biosensors (see e.g., Green and Kricka, 1984. Talanta31: 173-176; Blum, et al., 1989. J. Biolumin. Chemilumin. 4: 543-550).

The development of new reagents have made it possible to obtain stablelight emission proportional to the concentrations of ATP (see e.g.,Lundin, 1982. Applications of firefly luciferase In; Luminescent Assays(Raven Press, New York). With such stable light emission reagents, it ispossible to make endpoint assays and to calibrate each individual assayby addition of a known amount of ATP. In addition, a stablelight-emitting system also allows continuous monitoring ofATP-converting systems.

In a preferred embodiment, the ATP generating-ATP converting fusionprotein is attached to an affinity tag. The term “affinity tag” is usedherein to denote a peptide segment that can be attached to a polypeptideto provide for purification or detection of the polypeptide or providesites for attachment of the polypeptide to a substrate. In principal,any peptide or protein for which an antibody or other specific bindingagent is available can be used as an affinity tag. Affinity tags includea poly-histidine tract or a biotin carboxyl carrier protein (BCCP)domain, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith andJohnson, Gene 67:31, 1988), substance P, Flag.™.peptide (Hopp et al.,Biotechnology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.), streptavidin binding peptide, or other antigenic epitopeor binding domain. See, in general Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

As used herein, the term “poly-histidine tag,” when used in reference toa fusion protein refers to the presence of two to ten histidine residuesat either the amino- or carboxy-terminus of a protein of interest. Apoly-histidine tract of six to ten residues is preferred. Thepoly-histidine tract is also defined functionally as being a number ofconsecutive histidine residues added to the protein of interest whichallows the affinity purification of the resulting fusion protein on anickel-chelate or IDA column.

In some embodiments, the fusion protein has an orientation such that thesulfurylase polypeptide is N-terminal to the luciferase polypeptide. Inother embodiments, the luciferase polypeptide is N-terminal to thesulfurylase polypeptide. As used herein, the term sulfurylase-luciferasefusion protein refers to either of these orientations. The terms“amino-terminal” (N-terminal) and “carboxyl-terminal” (C-terminal) areused herein to denote positions within polypeptides and proteins. Wherethe context allows, these terms are used with reference to a particularsequence or portion of a polypeptide or protein to denote proximity orrelative position. For example, a certain sequence positionedcarboxyl-terminal to a reference sequence within a protein is locatedproximal to the carboxyl terminus of the reference sequence, but is notnecessarily at the carboxyl terminus of the complete protein.

The fusion protein of this invention can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor “sticky”-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). The two polypeptides of the fusionprotein can also be joined by a linker, such as a unique restrictionsite, which is engineered with specific primers during the cloningprocedure. In one embodiment, the sulfurylase and luciferasepolypeptides are joined by a linker, for example an ala-ala-ala linkerwhich is encoded by a Notl restriction site.

In one embodiment, the invention includes a recombinant polynucleotidethat comprises a coding sequence for a fusion protein having an ATPgenerating polypeptide sequence and an ATP converting polypeptidesequence. In a preferred embodiment, the recombinant polynucleotideencodes a sulfurylase-luciferase fusion protein. The term “recombinantDNA molecule” or “recombinant polynucleotide” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques. The term “recombinant protein”or “recombinant polypeptide” as used herein refers to a protein moleculewhich is expressed from a recombinant DNA molecule.

In one aspect, this invention discloses a sulfurylase-luciferase fusionprotein with an N-terminal hexahistidine tag and a BCCP tag. The nucleicacid sequence of the disclosed N-terminal hexahistidine-BCCPluciferase-sulfurylase gene (His6-BCCP L-S) gene is shown below:

His6-BCCP L-S Nucleotide Sequence:ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGGAAGCGCCAGCAGCA 60 (SEQ IDNO:3) GCGGAAATCAGTGGTCACATCGTACGTTCCCCGATGGTTGGTACTTTCTACCGCACCCCA 120AGCCCGGACGCAAAAGCGTTCATCGAAGTGGGTCAGAAAGTCAACGTGGGCGATACCCTG 180TGCATCGTTGAAGCCATGAAAATGATGAACCAGATCGAAGCGGACAAATCCGGTACCGTG 240AAAGCAATTCTGGTCGAAAGTGGACAACCGGTAGAATTTGACGAGCCGCTGGTCGTCATC 300GAGGGATCCGAGCTCGAGATCCAAATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCG 360CCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGA 420TACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACG 480TACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG 540AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTG 600TTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGT 660GAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGG 720TTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATG 780GATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTA 840CCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATT 900GCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCAT 960AGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATT 1020CCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACA 1080CTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTG 1140TTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTT 1200TCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATT 1260GCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCAT 1320CTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACA 1380CCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAG 1440GTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTC 1500AGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATT 1560GACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTC 1620TTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCGCT 1680GAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTT 1740CCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG 1800ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTG 1860CGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCA 1920AGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGTCCAAATTGGCGGCC 1980GCTATGCCTGCTCCTCACGGTGGTATTCTACAAGACTTGATTGCTAGAGATGCGTTAAAG 2040AAGAATGAATTGTTATCTGAAGCGCAATCTTCGGACATTTTAGTATGGAACTTGACTCCT 2100AGACAACTATGTGATATTGAATTGATTCTAAATGGTGGGTTTTCTCCTCTGACTGGGTTT 2160TTGAACGAAAACGATTACTCCTCTGTTGTTACAGATTCGAGATTAGCAGACGGCACATTG 2220TGGACCATCCCTATTACATTAGATGTTGATGAAGCATTTGCTAACCAAATTAAACCAGAC 2280ACAAGAATTGCCCTTTTCCAAGATGATGAAATTCCTATTGCTATACTTACTGTCCAGGAT 2340GTTTACAAGCCAAACAAAACTATCGAAGCCGAAAAAGTCTTCAGAGGTGACCCAGAACAT 2400CCAGCCATTAGCTATTTATTTAACGTTGCCGGTGATTATTACGTCGGCGGTTCTTTAGAA 2460GCGATTCAATTACCTCAACATTATGACTATCCAGGTTTGCGTAAGACACCTGCCCAACTA 2520AGACTTGAATTCCAATCAAGACAATGGGACCGTGTCGTAGCTTTCCAAACTCGTAATCCA 2580ATGCATAGAGCCCACAGGGAGTTGACTGTGAGAGCCGCCAGAGAAGCTAATGCTAAGGTG 2640CTGATCCATCCAGTTGTTGGACTAACCAAACCAGGTGATATAGACCATCACACTCGTGTT 2700CGTGTCTACCAGGAAATTATTAAGCGTTATCCTAATGGTATTGCTTTCTTATCCCTGTTG 2760CCATTAGCAATGAGAATGAGTGGTGATAGAGAAGCCGTATGGCATGCTATTATTAGAAAG 2820AATTATGGTGCCTCCCACTTCATTGTTGGTAGAGACCATGCGGGCCCAGGTAAGAACTCC 2880AAGGGTGTTGATTTCTACGGTCCATACGATGCTCAAGAATTGGTCGAATCCTACAAGCAT 2940GAACTGGACATTGAAGTTGTTCCATTCAGAATGGTCACTTATTTGCCAGACGAAGACCGT 3000TATGCTCCAATTGATCAAATTGACACCACAAAGACGAGAACCTTGAACATTTCAGGTACA 3060GAGTTGAGACGCCGTTTAAGAGTTGGTGGTGAGATTCCTGAATGGTTCTCATATCCTGAA 3120GTGGTTAAAATCCTAAGAGAATCCAACCCACCAAGACCAAAACAAGGTTTTTCAATTGTT 3180TTAGGTAATTCATTAACCGTTTCTCGTGAGCAATTATCCATTGCTTTGTTGTCAACATTC 3240TTGCAATTCGGTGGTGGCAGGTATTACAAGATCTTTGAACACAATAATAAGACAGAGTTA 3300CTATCTTTGATTCAAGATTTCATTGGTTCTGGTAGTGGACTAATTATTCCAAATCAATGG 3360GAAGATGACAAGGACTCTGTTGTTGGCAAGCAAAACGTTTACTTATTAGATACCTCAAGC 3420TCAGCCGATATTCAGCTAGAGTCAGCGGATGAACCTATTTCACATATTGTACAAAAAGTT 3480GTCCTATTCTTGGAAGACAATGGCTTTTTTGTATTTTAA 3519

The amino acid sequence of the disclosed His6-BCCP L-S polypeptide ispresented using the three letter amino acid code (SEQ ID NO:4).

His6-BCCP L-S Amino Acid Sequence Met Arg Gly Ser His His His His HisHis Gly Met Ala Ser Met Glu (SEQ ID NO:4)1               5                   10                   15 Ala Pro AlaAla Ala Glu Ile Ser Gly His Ile Val Arg Ser Pro Met            20                  25                  30 Val Gly Thr PheTyr Arg Thr Pro Ser Pro Asp Ala Lys Ala Phe Ile        35                  40                  45 Glu Val Gly Gln LysVal Asn Val Gly Asp Thr Leu Cys Ile Val Glu    50                  55                  60 Ala Met Lys Met Met AsnGln Ile Glu Ala Asp Lys Ser Gly Thr Val65                  70                  75                  80 Lys AlaIle Leu Val Glu Ser Gly Gln Pro Val Glu Phe Asp Glu Pro                85                  90                  95 Leu Val ValIle Glu Gly Ser Glu Leu Glu Ile Gln Met Glu Asp Ala            100                 105                 110 Lys Asn Ile LysLys Gly Pro Ala Pro Phe Tyr Pro Leu Glu Asp Gly        115                 120                 125 Thr Ala Gly Glu GlnLeu His Lys Ala Met Lys Arg Tyr Ala Leu Val    130                 135                 140 Pro Gly Thr Ile Ala PheThr Asp Ala His Ile Glu Val Asn Ile Thr145                 150                 155                 160 Tyr AlaGlu Tyr Phe Glu Met Ser Val Arg Leu Ala Glu Ala Met Lys                165                 170                 175 Arg Tyr GlyLeu Asn Thr Asn His Arg Ile Val Val Cys Ser Glu Asn            180                 185                 190 Ser Leu Gln PhePhe Met Pro Val Leu Gly Ala Leu Phe Ile Gly Val        195                 200                 205 Ala Val Ala Pro AlaAsn Asp Ile Tyr Asn Glu Arg Glu Leu Leu Asn    210                 215                 220 Ser Met Asn Ile Ser GlnPro Thr Val Val Phe Val Ser Lys Lys Gly225                 230                 235                 240 Leu GlnLys Ile Leu Asn Val Gln Lys Lys Leu Pro Ile Ile Gln Lys                245                 250                 255 Ile Ile IleMet Asp Ser Lys Thr Asp Tyr Gln Gly Phe Gln Ser Met            260                 265                 270 Tyr Thr Phe ValThr Ser His Leu Pro Pro Gly Phe Asn Glu Tyr Asp        275                 280                 285 Phe Val Pro Glu SerPhe Asp Arg Asp Lys Thr Ile Ala Leu Ile Met    290                 295                 300 Asn Ser Ser Gly Ser ThrGly Leu Pro Lys Gly Val Ala Leu Pro His305                 310                 315                 320 Arg ThrAla Cys Val Arg Phe Ser His Ala Arg Asp Pro Ile Phe Gly                325                 330                 335 Asn Gln IleIle Pro Asp Thr Ala Ile Leu Ser Val Val Pro Phe His            340                 345                 350 His Gly Phe GlyMet Phe Thr Thr Leu Gly Tyr Leu Ile Cys Gly Phe        355                 360                 365 Arg Val Val Leu MetTyr Arg Phe Glu Glu Gln Leu Phe Leu Arg Ser    370                 375                 380 Leu Gln Asp Tyr Lys IleGln Ser Ala Leu Leu Val Pro Thr Leu Phe385                 390                 395                 400 Ser PhePhe Ala Lys Ser Thr Leu Ile Asp Lys Tyr Asp Leu Ser Asn                405                 410                 415 Leu His GluIle Ala Ser Gly Gly Ala Pro Leu Ser Lys Glu Val Gly            420                 425                 430 Glu Ala Val AlaLys Arg Phe His Leu Pro Gly Ile Arg Gln Gly Tyr        435                 440                 445 Gly Leu Thr Glu ThrThr Ser Ala Ile Leu Ile Thr Pro Glu Gly Asp    450                 455                 460 Asp Lys Pro Gly Ala ValGly Lys Val Val Pro Phe Phe Glu Ala Lys465                 470                 475                 480 Val ValAsp Leu Asp Thr Gly Lys Thr Leu Gly Val Asn Gln Arg Gly                485                 490                 495 Glu Leu CysVal Arg Gly Pro Met Ile Met Ser Gly Tyr Val Asn Asn            500                 505                 510 Pro Glu Ala ThrAsn Ala Leu Ile Asp Lys Asp Gly Trp Leu His Ser        515                 520                 525 Gly Asp Ile Ala TyrTrp Asp Glu Asp Glu His Phe Phe Ile Val Asp    530                 535                 540 Arg Leu Lys Ser Leu IleLys Tyr Lys Gly Tyr Gln Val Ala Pro Ala545                 550                 555                 560 Glu LeuGlu Ser Ile Leu Leu Gln His Pro Asn Ile Phe Asp Ala Gly                565                 570                 575 Val Ala GlyLeu Pro Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala Val            580                 585                 590 Val Val Leu GluHis Gly Lys Thr Met Thr Glu Lys Glu Ile Val Asp        595                 600                 605 Tyr Val Ala Ser GlnVal Thr Thr Ala Lys Lys Leu Arg Gly Gly Val    610                 615                 620 Val Phe Val Asp Glu ValPro Lys Gly Leu Thr Gly Lys Leu Asp Ala625                630                 635                 640 Arg LysIle Arg Glu Ile Leu Ile Lys Ala Lys Lys Gly Gly Lys Ser                645                 650                 655 Lys Leu AlaAla Ala Met Pro Ala Pro His Gly Gly Ile Leu Gln Asp            660                 665                 670 Leu Ile Ala ArgAsp Ala Leu Lys Lys Asn Glu Leu Leu Ser Glu Ala            675                 680                 685 Gln Ser Ser AspIle Leu Val Trp Asn Leu Thr Pro Arg Gln Leu Cys        690                 695                 700 Asp Ile Glu Leu IleLeu Asn Gly Gly Phe Ser Pro Leu Thr Gly Phe    705                 710                 715 Leu Asn Glu Asn Asp TyrSer Ser Val Val Thr Asp Ser Arg Leu Ala720                 725                 730                 735 Asp GlyThr Leu Trp Thr Ile Pro Ile Thr Leu Asp Val Asp Glu Ala                740                 745                 750 Phe Ala AsnGln Ile Lys Pro Asp Thr Arg Ile Ala Leu Phe Gln Asp            755                 760                 765 Asp Glu Ile ProIle Ala Ile Leu Thr Val Gln Asp Val Tyr Lys Pro        770                 775                 780 Asn Lys Thr Ile GluAla Glu Lys Val Phe Arg Gly Asp Pro Glu His    785                 790                 795 Pro Ala Ile Ser Tyr LeuPhe Asn Val Ala Gly Asp Tyr Tyr Val Gly800                 805                 810                 815 Gly SerLeu Glu Ala Ile Gln Leu Pro Gln His Tyr Asp Tyr Pro Gly                820                 825                 830 Leu Arg LysThr Pro Ala Gln Leu Arg Leu Glu Phe Gln Ser Arg Gln            835                 840                 845 Trp Asp Arg ValVal Ala Phe Gln Thr Arg Asn Pro Met His Arg Ala        850                 855                 860 His Arg Glu Leu ThrVal Arg Ala Ala Arg Glu Ala Asn Ala Lys Val    865                 870                 875 Leu Ile His Pro Val ValGly Leu Thr Lys Pro Gly Asp Ile Asp His880                 885                 890                 895 His ThrArg Val Arg Val Tyr Gln Glu Ile Ile Lys Arg Tyr Pro Asn                900                 905                 910 Gly Ile AlaPhe Leu Ser Leu Leu Pro Leu Ala Met Arg Met Ser Gly            915                 920                 925 Asp Arg Glu AlaVal Trp His Ala Ile Ile Arg Lys Asn Tyr Gly Ala        930                 935                 940 Ser His Phe Ile ValGly Arg Asp His Ala Gly Pro Gly Lys Asn Ser    945                 950                 955 Lys Gly Val Asp Phe TyrGly Pro Tyr Asp Ala Gln Glu Leu Val Glu960                 965                 970                 975 Ser TyrLys His Glu Leu Asp Ile Glu Val Val Pro Phe Arg Met Val                980                 985                 990 Thr Tyr LeuPro Asp Glu Asp Arg Tyr Ala Pro Ile Asp Gln Ile Asp            995                1000                1005 Thr Thr Lys ThrArg Thr Leu Asn Ile Ser Gly Thr Glu Leu Arg Arg       1010                1015                1020 Arg Leu Arg Val GlyGly Glu Ile Pro Glu Trp Phe Ser Tyr Pro Glu   1025                1030                1035 Val Val Lys Ile Leu ArgGlu Ser Asn Pro Pro Arg Pro Lys Gln Gly1040               1045                1050                1055 Phe SerIle Val Leu Gly Asn Ser Leu Thr Val Ser Arg Glu Gln Leu               1060                1065                1070 Ser Ile AlaLeu Leu Ser Thr Phe Leu Gln Phe Gly Gly Gly Arg Tyr           1075                1080                1085 Tyr Lys Ile PheGlu His Asn Asn Lys Thr Glu Leu Leu Ser Leu Ile       1090                1095                1100 Gln Asp Phe Ile GlySer Gly Ser Gly Leu Ile Ile Pro Asn Gln Trp   1105                1110                1115 Glu Asp Asp Lys Asp SerVal Val Gly Lys Gln Asn Val Tyr Leu Leu1120               1125                1130                1135 Asp ThrSer Ser Ser Ala Asp Ile Gln Leu Glu Ser Ala Asp Glu Pro               1140                1145                1150 Ile Ser HisIle Val Gln Lys Val Val Leu Phe Leu Glu Asp Asn Gly           1155                1160                1165 Phe Phe Val Phe       1170

Accordingly, in one aspect, the invention provides for a fusion proteincomprising a thermostable sulfurylase joined to at least one affinitytag. The nucleic acid sequence of the disclosed N-terminalhexahistidine-BCCP Bst ATP Sulfurylase (His6-BCCP Bst Sulfurylase) geneis shown below:

His6-BCCP Bst Sulfurylase Nucleotide SequenceATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGGAAGCGCCAGCAGCA 60 (SEQ IDNO:5) GCGGAAATCAGTGGTCACATCGTACGTTCCCCGATGGTTGGTACTTTCTACCGCACCCCA 120AGCCCGGACGCAAAAGCGTTCATCGAAGTGGGTCAGAAAGTCAACGTGGGCGATACCCTG 180TGCATCGTTGAAGCCATGAAAATGATGAACCAGATCGAAGCGGACAAATCCGGTACCGTG 240AAAGCAATTCTGGTCGAAAGTGGACAACCGGTAGAATTTGACGAGCCGCTGGTCGTCATC 300GAGGGATCCGAGCTCGAGATCTGCAGCATGAGCGTAAGCATCCCGCATGGCGGCACATTG 360ATCAACCGTTGGAATCCGGATTACCCAATCGATGAAGCAACGAAAACGATCGAGCTGTCC 420AAAGCCGAACTAAGCGACCTTGAGCTGATCGGCACAGGCGCCTACAGCCCGCTCACCGGG 480TTTTTAACGAAAGCCGATTACGATGCGGTCGTAGAAACGATGCGCCTCGCTGATGGCACT 540GTCTGGAGCATTCCGATCACGCTGGCGGTGACGGAAGAAAAAGCGAGTGAACTCACTGTC 600GGCGACAAAGCGAAACTCGTTTATGGCGGCGACGTCTACGGCGTCATTGAAATCGCCGAT 660ATTTACCGCCCGGATAAAACGAAAGAAGCCAAGCTCGTCTATAAAACCGATGAACTCGCT 720CACCCGGGCGTGCGCAAGCTGTTTGAAAAACCAGATGTGTACGTCGGCGGAGCGGTTACG 780CTCGTCAAACGGACCGACAAAGGCCAGTTTGCTCCGTTTTATTTCGATCCGGCCGAAACG 840CGGAAACGATTTGCCGAACTCGGCTGGAATACCGTCGTCGGCTTCCAAACACGCAACCCG 900GTTCACCGCGCCCATGAATACATTCAAAAATGCGCGCTTGAAATCGTGGACGGCTTGTTT 960TTAAACCCGCTCGTCGGCGAAACGAAAGCGGACGATATTCCGGCCGACATCCGGATGGAA 1020AGCTATCAAGTGCTGCTGGAAAACTATTATCCGAAAGACCGCGTTTTCTTGGGCGTCTTC 1080CAAGCTGCGATGCGCTATGCCGGTCCGCGCGAAGCGATTTTCCATGCCATGGTGCGGAAA 1140AACTTCGGCTGCACGCACTTCATCGTCGGCCGCGACCATGCGGGCGTCGGCAACTATTAC 1200GGCACGTATGATGCGCAAAAAATCTTCTCGAACTTTACAGCCGAAGAGCTTGGCATTACA 1260CCGCTCTTTTTCGAACACAGCTTTTATTGCACGAAATGCGAAGGCATGGCATCGACGAAA 1320ACATGCCCGCACGACGCACAATATCACGTTGTCCTTTCTGGCACGAAAGTCCGTGAAATG 1380TTGCGTAACGGCCAAGTGCCGCCGAGCACATTCAGCCGTCCGGAAGTGGCCGCCGTTTTG 1440ATCAAAGGGCTGCAAGAACGCGAAACGGTCGCCCCGTCAGCGCGCTAA 1488

The amino acid sequence of the His6-BCCP Bst Sulfurylase polypeptide ispresented using the three letter amino acid code in Table 6 (SEQ IDNO:6).

His6-BCCP Bst Sulfurylase Amino Acid Sequence Met Arg Gly Ser His HisHis His His His Gly Met Ala Ser Met Glu (SEQ ID NO:6)1               5                   10                  15 Ala Pro AlaAla Ala Glu Ile Ser Gly His Ile Val Arg Ser Pro Met            20                  25                  30 Val Gly Thr PheTyr Arg Thr Pro Ser Pro Asp Ala Lys Ala Phe Ile        35                  40                  45 Glu Val Gly Gln LysVal Asn Val Gly Asp Thr Leu Cys Ile Val Glu    50                  55                  60 Ala Met Lys Met Met AsnGln Ile Glu Ala Asp Lys Ser Gly Thr Val65                   70                  75                  80 Lys AlaIle Leu Val Glu Ser Gly Gln Pro Val Glu Phe Asp Glu Pro                85                  90                  95 Leu Val ValIle Glu Gly Ser Glu Leu Glu Ile Cys Ser Met Ser Val            100                 105                 110 Ser Ile Pro HisGly Gly Thr Leu Ile Asn Arg Trp Asn Pro Asp Tyr        115                 120                 125 Pro Ile Asp Glu AlaThr Lys Thr Ile Glu Leu Ser Lys Ala Glu Leu    130                 135                 140 Ser Asp Leu Glu Leu IleGly Thr Gly Ala Tyr Ser Pro Leu Thr Gly145                 150                 155                 160 Phe LeuThr Lys Ala Asp Tyr Asp Ala Val Val Glu Thr Met Arg Leu                165                 170                 175 Ala Asp GlyThr Val Trp Ser Ile Pro Ile Thr Leu Ala Val Thr Glu            180                 185                 190 Glu Lys Ala SerGlu Leu Thr Val Gly Asp Lys Ala Lys Leu Val Tyr        195                 200                 205 Gly Gly Asp Val TyrGly Val Ile Glu Ile Ala Asp Ile Tyr Arg Pro    210                 215                 220 Asp Lys Thr Lys Glu AlaLys Leu Val Tyr Lys Thr Asp Glu Leu Ala225                 230                 235                 240 His ProGly Val Arg Lys Leu Phe Glu Lys Pro Asp Val Tyr Val Gly                245                 250                 255 Gly Ala ValThr Leu Val Lys Arg Thr Asp Lys Gly Gln Phe Ala Pro            260                 265                 270 Phe Tyr Phe AspPro Ala Glu Thr Arg Lys Arg Phe Ala Glu Leu Gly        275                 280                 285 Trp Asn Thr Val ValGly Phe Gln Thr Arg Asn Pro Val His Arg Ala    290                 295                 300 His Glu Tyr Ile Gln LysCys Ala Leu Glu Ile Val Asp Gly Leu Phe305                 310                 315                 320 Leu AsnPro Leu Val Gly Glu Thr Lys Ala Asp Asp Ile Pro Ala Asp                325                 330                 335 Ile Arg MetGlu Ser Tyr Gln Val Leu Leu Glu Asn Tyr Tyr Pro Lys            340                 345                 350 Asp Arg Val PheLeu Gly Val Phe Gln Ala Ala Met Arg Tyr Ala Gly        355                 360                 365 Pro Arg Glu Ala IlePhe His Ala Met Val Arg Lys Asn Phe Gly Cys    370                 375                 380 Thr His Phe Ile Val GlyArg Asp His Ala Gly Val Gly Asn Tyr Tyr385                 390                 395                 400 Gly ThrTyr Asp Ala Gln Lys Ile Phe Ser Asn Phe Thr Ala Glu Glu                405                 410                 415 Leu Gly IleThr Pro Leu Phe Phe Glu His Ser Phe Tyr Cys Thr Lys            420                 425                 430 Cys Glu Gly MetAla Ser Thr Lys Thr Cys Pro His Asp Ala Gln Tyr        435                 440                 445 His Val Val Leu SerGly Thr Lys Val Arg Glu Met Leu Arg Asn Gly    450                 455                 460 Gln Val Pro Pro Ser ThrPhe Ser Arg Pro Glu Val Ala Ala Val Leu465                 470                 475                 480 Ile LysGly Leu Gln Glu Arg Glu Thr Val Ala Pro Ser Ala Arg                485                 490                 495

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an ATP generatingpolypeptide and an ATP converting polypeptide, or derivatives,fragments, analogs or homologs thereof. As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid in a form suitable for expression of the nucleic acid in a hostcell, which means that the recombinant expression vectors include one ormore regulatory sequences, selected on the basis of the host cells to beused for expression, that is operatively linked to the nucleic acidsequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory sequence(s) in a manner that allowsfor expression of the nucleotide sequence (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produce afusion protein.

The recombinant expression vectors of the invention can be designed forexpression of the fusion protein in prokaryotic or eukaryotic cells. Forexample, a sulfurylase-luciferase fusion protein can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression. Fusion vectors add a number of amino acids toa protein encoded therein, usually to the amino terminus of therecombinant protein. Such fusion vectors typically serve three purposes:(1) to increase expression of recombinant protein; (2) to increase thesolubility of the recombinant protein; and (3) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

In another embodiment, the ATP generating-ATP converting fusion proteinexpression vector is a yeast expression vector. Examples of vectors forexpression in yeast S. cerivisae include pYepSec1 (Baldari, et al.,(1987) EMBO J 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp,San Diego, Calif.).

Alternatively, the fusion protein can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. The invention alsoincludes a kit comprising a sulfurylase-luciferase fusion proteinexpression vector.

A host cell can be any prokaryotic or eukaryotic cell. For example, thesulfurylase-luciferase fusion protein can be expressed in bacterialcells such as E. coli, insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding ORFX or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) the fusionprotein. Accordingly, the invention further provides methods forproducing the fusion protein using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding thefusion protein has been introduced) in a suitable medium such that thefusion protein is produced. In another embodiment, the method furthercomprises isolating the fusion protein from the medium or the host cell.

The invention also includes a fusion protein bound to a mobile support.In a preferred embodiment, the fusion gene is a sulfurylase-luciferasefusion gene. In another embodiment, the mobile support is bound tostrepavidin. The mobile support could be a bead or optical fiber. In apreferred embodiment, the bead is a nickel-agarose bead or aMPG-Streptavidin bead. In one embodiment, the sulfurylase-luciferasefusion protein is bound to the beads in a 1:3 ratio of protein to bead.It can be attached to the solid support via a covalent or non-covalentinteraction. In general, any linkage recognized in the art can be used.Examples of such linkages common in the art include any suitable metal(e.g., Co²⁺, Ni²⁺)-hexahistidine complex, a biotin binding protein,e.g., NEUTRAVIDIN™ modified avidin (Pierce Chemicals, Rockford, Ill.),streptavidin/biotin, avidin/biotin, glutathione S-transferase(GST)/glutathione, monoclonal antibody/antigen, and maltose bindingprotein/maltose, and pluronic coupling technologies. Samples containingthe appropriate tag are incubated with the sensitized substrate so thatzero, one, or multiple molecules attach at each sensitized site.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in denovo fatty acid biosynthesis. It belongs to a group of carboxylases thatuse biotin as cofactor and bicarbonate as a source of the carboxylgroup. There are two types of ACCase: prokaryotic ACCase (e.g., E. coli,P. aeruginosa, Anabaena, Synechococcus and probably pea chloroplast) inwhich the three functional domains: biotin carboxylase (BC), biotincarboxyl carrier protein (BCCP) and carboxyltransferase (CT) are locatedon separable subunits and eukaryotic ACCase (e.g., rat, chicken, yeast,diatom and wheat) in which all the domains are located on one largepolypeptide. It is known that a BCCP as a subunit of acetyl CoAcarboxylase from E. coli is biotinated at the Lys residue at the122-position by the action of biotin holoenzyme synthetase in E. coli(Journal of Biological Chemistry, 263, 6461 (1988)). In a preferredembodiment of this invention, the fusion protein is bound to a BCCPdomain which is then utilized for binding avidins; therefore, it canbind to a streptavidin mobile support. One biotin-(strept-)avidin-basedanchoring method uses a thin layer of a photoactivatable biotin analogdried onto a solid surface. (Hengsakul and Cass, 1996. BioconjugateChem. 7: 249-254). The biotin analog is then exposed to white lightthrough a mask, so as to create defined areas of activated biotin.Avidin (or streptavidin) is then added and allowed to bind to theactivated biotin. The avidin possesses free biotin binding sites whichcan be utilized to “anchor” the biotinylated proteins through abiotin-(strept-)avidin linkage.

Alternatively, the fusion protein can be attached to the solid supportwith a biotin derivative possessing a photo-removable protecting group.This moiety is covalently bound to bovine serum albumin (BSA), which isattached to the solid support, e.g., a glass surface. See Pirrung andHuang, 1996. Bioconjugate Chem. 7: 317-321. A mask is then used tocreate activated biotin within the defined irradiated areas. Avidin maythen be localized to the irradiated area, with a biotinylatedsulfurylase-luciferase fusion protein subsequently attached through aBSA-biotin-avidin-biotin link.

Another method of attachment is with the use of a pluronics basedattachment. Pluronics attach to hydrophobic surfaces by virtue of thereaction between the hydrophobic surface and the polypropylene oxide.The remaining polyethylene oxide groups extend off the surface, therebycreating a hydrophilic environment. Nitrilotriacetic acid (NTA) can beconjugated to the terminal ends of the polyethylene oxide chains toallow for hexahistidine tagged proteins to be attached.

This invention provides methods of sequencing which utilize and ATPgenerating polypeptide-ATP converting polypeptide fusion protein fordetection. In a preferred embodiment, the nucleotide sequence of thesequencing product is determined by measuring inorganic pyrophosphate(PPi) liberated from a nucleotide triphosphate (dNTP) as the dNMP isincorporated into an extended sequence primer. This method of sequencingis termed Pyrosequencing•technology (PyroSequencing AB, Stockholm,Sweden). It can be performed in solution (liquid phase) or as a solidphase technique. Various sequencing methods, including PPi sequencingmethods, are described in, e.g., WO9813523A1, Ronaghi, et al., 1996.Anal. Biochem. 242: 84-89, and Ronaghi, et al., 1998. Science 281:363-365 (1998), U.S. Pat. No. 6,274,320 and the patent application U.S.Ser. No. 10/104,280 which was filed on Mar. 21, 2001 (21465-501CIP3).These disclosures of sequencing are incorporated herein in theirentirety, by reference.

Pyrophosphate released under these conditions can be detectedenzymatically (e.g., by the generation of light in theluciferase-luciferin reaction). Such methods enable a nucleotide to beidentified in a given target position, and the DNA to be sequencedsimply and rapidly while avoiding the need for electrophoresis and theuse of potentially dangerous radiolabels.

The invention also provides a method for sequencing nucleic acids whichgenerally comprises (a) providing one or more nucleic acid anchorprimers and a plurality of single-stranded circular nucleic acidtemplates disposed within a plurality of reaction chambers or cavities;(b) annealing an effective amount of the nucleic acid anchor primer toat least one of the single-stranded circular templates to yield a primedanchor primer-circular template complex; (c) combining the primed anchorprimer-circular template complex with a polymerase to form an extendedanchor primer covalently linked to multiple copies of a nucleic acidcomplementary to the circular nucleic acid template; (d) annealing aneffective amount of a sequencing primer to one or more copies of saidcovalently linked complementary nucleic acid; (e) extending thesequencing primer with a polymerase and a predetermined nucleotidetriphosphate to yield a sequencing product and, if the predeterminednucleotide triphosphate is incorporated onto the 3′ end of saidsequencing primer, a sequencing reaction byproduct; and (f) identifyingthe PPi sequencing reaction byproduct with the use of an ATP generatingpolypeptide-ATP converting polypeptide fusion protein, therebydetermining the sequence of the nucleic acid. In one embodiment, a dATPor ddATP analogue is used in place of deoxy- or dideoxy adenosinetriphosphate. This analogue is capable of acting as a substrate for apolymerase but incapable of acting as a substrate for a PPi-detectionenzyme. This method can be carried out in separate parallel commonreactions in an aqueous environment.

In another aspect, the invention includes a method of determining thebase sequence of a plurality of nucleotides on an array, which generallycomprises (a) providing a plurality of sample DNAs, each disposed withina plurality of cavities on a planar surface; (b) adding an activatednucleotide 5′-triphosphate precursor of one known nitrogenous base to areaction mixture in each reaction chamber, each reaction mixturecomprising a template-directed nucleotide polymerase and asingle-stranded polynucleotide template hybridized to a complementaryoligonucleotide primer strand at least one nucleotide residue shorterthan the templates to form at least one unpaired nucleotide residue ineach template at the 3′-end of the primer strand, under reactionconditions which allow incorporation of the activated nucleoside5′-triphosphate precursor onto the 3′-end of the primer strands,provided the nitrogenous base of the activated nucleoside5′-triphosphate precursor is complementary to the nitrogenous base ofthe unpaired nucleotide residue of the templates; (c) utilizing an ATPgenerating polypeptide-ATP converting polypeptide fusion protein todetect whether or not the nucleoside 5′-triphosphate precursor wasincorporated into the primer strands in which incorporation of thenucleoside 5′-triphosphate precursor indicates that the unpairednucleotide residue of the template has a nitrogenous base compositionthat is complementary to that of the incorporated nucleoside5′-triphosphate precursor; and (d) sequentially repeating steps (b) and(c), wherein each sequential repetition adds and, detects theincorporation of one type of activated nucleoside 5′-triphosphateprecursor of known nitrogenous base composition; and (e) determining thebase sequence of the unpaired nucleotide residues of the template ineach reaction chamber from the sequence of incorporation of saidnucleoside precursors.

The anchor primers of the invention generally comprise a stalk regionand at least one adaptor region. In a preferred embodiment the anchorprimer contains at least two contiguous adapter regions. The stalkregion is present at the 5′ end of the anchor primer and includes aregion of nucleotides for attaching the anchor primer to the solidsubstrate.

The adaptor region(s) comprise nucleotide sequences that hybridize to acomplementary sequence present in one or more members of a population ofnucleic acid sequences. In some embodiments, the anchor primer includestwo adjoining adaptor regions, which hybridize to complementary regionsligated to separate ends of a target nucleic acid sequence. Inadditional embodiments, the adapter regions in the anchor primers arecomplementary to non-contiguous regions of sequence present in a secondnucleic acid sequence. Each adapter region, for example, can behomologous to each terminus of a fragment produced by digestion with oneor more restriction endonucleases. The fragment can include, e.g., asequence known or suspected to contain a sequence polymorphism.Additionally, the anchor primer may contain two adapter regions that arehomologous to a gapped region of a target nucleic acid sequence, i.e.,one that is non-contiguous because of a deletion of one or morenucleotides. When adapter regions having these sequences are used, analigning oligonucleotide corresponding to the gapped sequence may beannealed to the anchor primer along with a population of templatenucleic acid molecules.

The anchor primer may optionally contain additional elements such as oneor more restriction enzyme recognition sites, RNA polymerase bindingsites, e.g., a T7 promoter site, or sequences present in identified DNAsequences, e.g., sequences present in known genes. The adapter region(s)may also include sequences known to flank sequence polymorphisms.Sequence polymorphisms include nucleotide substitutions, insertions,deletions, or other rearrangements which result in a sequence differencebetween two otherwise identical nucleic acid sequences. An example of asequence polymorphism is a single nucleotide polymorphism (SNP).

In general, any nucleic acid capable of base-pairing can be used as ananchor primer. In some embodiments, the anchor primer is anoligonucleotide. As utilized herein the term oligonucleotide includeslinear oligomers of natural or modified monomers or linkages, e.g.,deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptidenucleic acids (PNAs), and the like, that are capable of specificallybinding to a target polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions. These types of interactions caninclude, e.g., Watson-Crick type of base-pairing, base stacking,Hoogsteen or reverse-Hoogsteen types of base-pairing, or the like.Generally, the monomers are linked by phosphodiester bonds, or analogsthereof, to form oligonucleotides ranging in size from, e.g., 3-200,8-150, 10-100, 20-80, or 25-50 monomeric units. Whenever anoligonucleotide is represented by a sequence of letters, it isunderstood that the nucleotides are oriented in the 5′→3′ direction,from left-to-right, and that the letter “A” donates deoxyadenosine, theletter “T” denotes thymidine, the letter “C” denotes deoxycytosine, andthe letter “G” denotes deoxyguanosine, unless otherwise noted herein.The oligonucleotides of the present invention can include non-naturalnucleotide analogs. However, where, for example, processing by enzymesis required, or the like, oligonucleotides comprising naturallyoccurring nucleotides are generally required for maintenance ofbiological function.

Anchor primers are linked to the solid substrate at the sensitizedsites. They can be linked by the same method of linkage as described forthe fusion protein to the solid support. A region of a solid substratecontaining a linked primer is referred to herein as an anchor pad. Thus,by specifying the sensitized states on the solid support, it is possibleto form an array or matrix of anchor pads. The anchor pads can be, e.g.,small diameter spots etched at evenly spaced intervals on the solidsupport. The anchor pads can be located at the bottoms of thecavitations or wells if the substrate has been cavitated, etched, orotherwise micromachined as discussed above.

In one embodiment, the anchor primer is linked to a particle. The anchorprimer can be linked to the particle prior to formation of the extendedanchor primer or after formation of the extended anchor primer.

Each sensitized site on a solid support is potentially capable ofattaching multiple anchor primers. Thus, each anchor pad may include oneor more anchor primers. It is preferable to maximize the number of padsthat have only a single productive reaction center (e.g., the number ofpads that, after the extension reaction, have only a single sequenceextended from the anchor primer). This can be accomplished by techniqueswhich include, but are not limited to: (i) varying the dilution ofbiotinylated anchor primers that are washed over the surface; (ii)varying the incubation time that the biotinylated primers are in contactwith the avidin surface; (iii) varying the concentration of open- orclosed-circular template so that, on average, only one primer on eachpad is extended to generate the sequencing template; or (iv) reducingthe size of the anchor pad to approach single-molecule dimensions (<1μm) such that binding of one anchor inhibits or blocks the binding ofanother anchor (e.g. by photoactivation of a small spot); or (v)reducing the size of the anchor pad such that binding of one circulartemplate inhibits or blocks the binding of a second circular template.

In some embodiments, each individual pad contains just one linked anchorprimer. Pads having only one anchor primer can be made by performinglimiting dilutions of a selected anchor primer on to the solid supportsuch that, on average, only one anchor primer is deposited on each pad.The concentration of anchor primer to be applied to a pad can becalculated utilizing, for example, a Poisson distribution model.

In order to maximize the number of reaction pads that contain a singleanchor primer, a series of dilution experiments are performed in which arange of anchor primer concentrations or circular templateconcentrations are varied. For highly dilute concentrations of primers,primers and circular templates binding to the same pad will beindependent of each other, and a Poisson distribution will characterizethe number of anchor primers extended on any one pad. Although therewill be variability in the number of primers that are actually extended,a maximum of 37% of the pads will have a single extended anchor primer(the number of pads with a single anchor oligonucleotide).

In other embodiments multiple anchor primers are attached to any oneindividual pad in an array. Limiting dilutions of a plurality ofcircular nucleic acid templates (described in more detail below) may behybridized to the anchor primers so immobilized such that, on average,only one primer on each pad is hybridized to a nucleic acid template.Library concentrations to be used may be calculated utilizing, forexample, limiting dilutions and a Poisson distribution model.

The nucleic acid templates that can be sequenced according to theinvention, e.g., a nucleic acid library, in general can include opencircular or closed circular nucleic acid molecules. A “closed circle” isa covalently closed circular nucleic acid molecule, e.g., a circular DNAor RNA molecule. An “open circle” is a linear single-stranded nucleicacid molecule having a 5′ phosphate group and a 3′ hydroxyl group. Inone embodiment, the single stranded nucleic acid contains at least 100copies of nucleic acid sequence, each copy covalently linked end to end.In some embodiments, the open circle is formed in situ from a lineardouble-stranded nucleic acid molecule. The ends of a given open circlenucleic acid molecule can be ligated by DNA ligase. Sequences at the 5′and 3′ ends of the open circle molecule are complementary to two regionsof adjacent nucleotides in a second nucleic acid molecule, e.g., anadapter region of an anchor primer, or to two regions that are nearlyadjoining in a second DNA molecule. Thus, the ends of the open-circlemolecule can be ligated using DNA ligase, or extended by DNA polymerasein a gap-filling reaction. Open circles are described in detail inLizardi, U.S. Pat. No. 5,854,033. An open circle can be converted to aclosed circle in the presence of a DNA ligase (for DNA) or RNA ligasefollowing, e.g., annealing of the open circle to an anchor primer.

If desired, nucleic acid templates can be provided as padlock probes.Padlock probes are linear oligonucleotides that includetarget-complementary sequences located at each end, and which areseparated by a linker sequence. The linkers can be ligated to ends ofmembers of a library of nucleic acid sequences that have been, e.g.,physically sheared or digested with restriction endonucleases. Uponhybridization to a target-sequence, the 5′- and 3′-terminal regions ofthese linear oligonucleotides are brought in juxtaposition. Thisjuxtaposition allows the two probe segments (if properly hybridized) tobe covalently-bound by enzymatic ligation (e.g., with T4 DNA ligase),thus converting the probes to circularly-closed molecules which arecatenated to the specific target sequences (see e.g., Nilsson, et al.,1994. Science 265: 2085-2088). The resulting probes are suitable for thesimultaneous analysis of many gene sequences both due to theirspecificity and selectivity for gene sequence variants (see e.g.,Lizardi, et al., 1998. Nat. Genet. 19: 225-232; Nilsson, et al., 1997.Nat. Genet. 16: 252-255) and due to the fact that the resulting reactionproducts remain localized to the specific target sequences. Moreover,intramolecular ligation of many different probes is expected to be lesssusceptible to non-specific cross-reactivity than multiplex PCR-basedmethodologies where non-cognate pairs of primers can give rise toirrelevant amplification products (see e.g., Landegren and Nilsson,1997. Ann. Med. 29: 585-590).

A starting library can be constructed comprising either single-strandedor double-stranded nucleic acid molecules, provided that the nucleicacid sequence includes a region that, if present in the library, isavailable for annealing, or can be made available for annealing, to ananchor primer sequence. For example, when used as a template for rollingcircle amplification, a region of a double-stranded template needs to beat least transiently single-stranded in order to act as a template forextension of the anchor primer.

Library templates can include multiple elements, including, but notlimited to, one or more regions that are complementary to the anchorprimer. For example, the template libraries may include a regioncomplementary to a sequencing primer, a control nucleotide region, andan insert sequence comprised of the sequencing template to besubsequently characterized. As is explained in more detail below, thecontrol nucleotide region is used to calibrate the relationship betweenthe amount of byproduct and the number of nucleotides incorporated. Asutilized herein the term “complement” refers to nucleotide sequencesthat are able to hybridize to a specific nucleotide sequence to form amatched duplex.

In one embodiment, a library template includes: (i) two distinct regionsthat are complementary to the anchor primer, (ii) one region homologousto the sequencing primer, (iii) one optional control nucleotide region,(iv) an insert sequence of, e.g., 30-500, 50-200, or 60-100 nucleotides,that is to be sequenced. The template can, of course, include two,three, or all four of these features.

The template nucleic acid can be constructed from any source of nucleicacid, e.g., any cell, tissue, or organism, and can be generated by anyart-recognized method. Suitable methods include, e.g., sonication ofgenomic DNA and digestion with one or more restriction endonucleases(RE) to generate fragments of a desired range of lengths from an initialpopulation of nucleic acid molecules. Preferably, one or more of therestriction enzymes have distinct four-base recognition sequences.Examples of such enzymes include, e.g., Sau3A1, MspI, and TaqI.Preferably, the enzymes are used in conjunction with anchor primershaving regions containing recognition sequences for the correspondingrestriction enzymes. In some embodiments, one or both of the adapterregions of the anchor primers contain additional sequences adjoiningknown restriction enzyme recognition sequences, thereby allowing forcapture or annealing to the anchor primer of specific restrictionfragments of interest to the anchor primer. In other embodiments, therestriction enzyme is used with a type IIS restriction enzyme.

Alternatively, template libraries can be made by generating acomplementary DNA (cDNA) library from RNA, e.g., messenger RNA (mRNA).The cDNA library can, if desired, be further processed with restrictionendonucleases to obtain a 3′ end characteristic of a specific RNA,internal fragments, or fragments including the 3 end of the isolatedRNA. Adapter regions in the anchor primer may be complementary to asequence of interest that is thought to occur in the template library,e.g., a known or suspected sequence polymorphism within a fragmentgenerated by endonuclease digestion.

In one embodiment, an indexing oligonucleotide can be attached tomembers of a template library to allow for subsequent correlation of atemplate nucleic acid with a population of nucleic acids from which thetemplate nucleic acid is derived. For example, one or more samples of astarting DNA population can be fragmented separately using any of thepreviously disclosed methods (e.g., restriction digestion, sonication).An indexing oligonucleotide sequence specific for each sample isattached to, e.g., ligated to, the termini of members of the fragmentedpopulation. The indexing oligonucleotide can act as a region forcircularization, amplification and, optionally, sequencing, whichpermits it to be used to index, or code, a nucleic acid so as toidentify the starting sample from which it is derived.

Distinct template libraries made with a plurality of distinguishableindexing primers can be mixed together for subsequent reactions.Determining the sequence of the member of the library allows for theidentification of a sequence corresponding to the indexingoligonucleotide. Based on this information, the origin of any givenfragment can be inferred.

Libraries of nucleic acids are annealed to anchor primer sequences usingrecognized techniques (see, e.g., Hatch, et al., 1999. Genet. Anal.Biomol. Engineer. 15: 35-40; Kool, U.S. Pat. No. 5,714,320 and Lizardi,U.S. Pat. No. 5,854,033). In general, any procedure for annealing theanchor primers to the template nucleic acid sequences is suitable aslong as it results in formation of specific, i.e., perfect or nearlyperfect, complementarity between the adapter region or regions in theanchor primer sequence and a sequence present in the template library.

A number of in vitro nucleic acid amplification techniques may beutilized to extend the anchor primer sequence. The size of the amplifiedDNA preferably is smaller than the size of the anchor pad and alsosmaller than the distance between anchor pads.

The amplification is typically performed in the presence of apolymerase, e.g., a DNA or RNA-directed DNA polymerase, and one, two,three, or four types of nucleotide triphosphates, and, optionally,auxiliary binding proteins. In general, any polymerase capable ofextending a primed 3′-OH group can be used a long as it lacks a 3′ to 5′exonuclease activity. Suitable polymerases include, e.g., the DNApolymerases from Bacillus stearothermophilus, Thermus acquaticus,Pyrococcus furiosis, Thermococcus litoralis, and Thermus thermophilus,bacteriophage T4 and T7, and the E. coli DNA polymerase I Klenowfragment. Suitable RNA-directed DNA polymerases include, e.g., thereverse transcriptase from the Avian Myeloblastosis Virus, the reversetranscriptase from the Moloney Murine Leukemia Virus, and the reversetranscriptase from the Human Immunodeficiency Virus-I.

A number of in vitro nucleic acid amplification techniques have beendescribed. These amplification methodologies may be differentiated intothose methods: (i) which require temperature cycling—polymerase chainreaction (PCR) (see e.g., Saiki, et al., 1995. Science 230: 1350-1354),ligase chain reaction (see e.g., Barany, 1991. Proc. Natl. Acad. Sci.USA 88: 189-193; Barringer, et al., 1990. Gene 89: 117-122) andtranscription-based amplification (see e.g., Kwoh, et al, 1989. Proc.Natl. Acad. Sci. USA 86: 1173-1177) and (ii) isothermal amplificationsystems—self-sustaining, sequence replication (see e.g., Guatelli, etal., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878); the Qβ replicasesystem (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202);strand displacement amplification Nucleic Acids Res. Apr. 11,1992;20(7):1691-6.; and the methods described in PNAS Jan. 1,1992;89(1):392-6; and NASBA J Virol Methods. 1991 December;35(3):273-86.

Isothermal amplification also includes rolling circle-basedamplification (RCA). RCA is discussed in, e.g., Kool, U.S. Pat. No.5,714,320 and Lizardi, U.S. Pat. No. 5,854,033; Hatch, et al., 1999.Genet. Anal. Biomol. Engineer. 15: 35-40. The result of the RCA is asingle DNA strand extended from the 3′ terminus of the anchor primer(and thus is linked to the solid support matrix) and including aconcatamer containing multiple copies of the circular template annealedto a primer sequence. Typically, 1,000 to 10,000 or more copies ofcircular templates, each having a size of, e.g., approximately 30-500,50-200, or 60-100 nucleotides size range, can be obtained with RCA.

In vivo, RCR is utilized in several biological systems. For example, thegenome of several bacteriophage are single-stranded, circular DNA.During replication, the circular DNA is initially converted to a duplexform, which is then replicated by the aforementioned rolling-circlereplication mechanism. The displaced terminus generates a series ofgenomic units that can be cleaved and inserted into the phage particles.Additionally, the displaced single-strand of a rolling-circle can beconverted to duplex DNA by synthesis of a complementary DNA strand. Thissynthesis can be used to generate the concatemeric duplex moleculesrequired for the maturation of certain phage DNAs. For example, thisprovides the principle pathway by which λ bacteriophage matures. RCR isalso used in vivo to generate amplified rDNA in Xenopus oocytes, andthis fact may help explain why the amplified rDNA is comprised of alarge number of identical repeating units. In this case, a singlegenomic repeating unit is converted into a rolling-circle. The displacedterminus is then converted into duplex DNA which is subsequently cleavedfrom the circle so that the two termini can be ligated together so as togenerate the amplified circle of rDNA.

Through the use of the RCA reaction, a strand may be generated whichrepresents many tandem copies of the complement to the circularizedmolecule. For example, RCA has recently been utilized to obtain anisothermal cascade amplification reaction of circularized padlock probesin vitro in order to detect single-copy genes in human genomic DNAsamples (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232). Inaddition, RCA has also been utilized to detect single DNA molecules in asolid phase-based assay, although difficulties arose when this techniquewas applied to in situ hybridization (see Lizardi, et al., 1998. Nat.Genet. 19: 225-232).

If desired, RCA can be performed at elevated temperatures, e.g., attemperatures greater than 37° C., 42° C., 45° C., 50° C., 60° C., or 70°C. In addition initially at a lower temperature, e.g., room temperature,and then shifted to an elevated temperature. Elevated temperature RCA ispreferably performed with thermostable nucleic acid polymerases and withprimers that can anneal stably and with specificity at elevatedtemperatures.

RCA can also be performed with non-naturally occurring oligonucleotides,e.g., peptide nucleic acids. Further, RCA can be performed in thepresence of auxiliary proteins such as single-stranded binding proteins.

The development of a method of amplifying short DNA molecules which havebeen immobilized to a solid support, termed RCA has been recentlydescribed in the literature (see e.g., Hatch, et al., 1999. Genet. Anal.Biomol. Engineer. 15: 35-40; Zhang, et al., 1998. Gene 211: 277-85;Baner, et al., 1998. Nucl. Acids Res. 26: 5073-5078; Liu, et al., 1995.J. Am. Chem. Soc. 118: 1587-1594; Fire and Xu, 1995. Proc. Natl. Acad.Sci. USA 92: 4641-4645; Nilsson, et al., 1994. Science 265: 2085-2088).RCA targets specific DNA sequences through hybridization and a DNAligase reaction. The circular product is then subsequently used as atemplate in a rolling circle replication reaction.

RCA driven by DNA polymerase can replicate circularized oligonucleotideprobes with either linear or geometric kinetics under isothermalconditions. In the presence of two primers (one hybridizing to the+strand, and the other, to the −strand of DNA), a complex pattern of DNAstrand displacement ensues which possesses the ability to generate 1×10⁹or more copies of each circle in a short period of time (i.e., less-than90 minutes), enabling the detection of single-point mutations within thehuman genome. Using a single primer, RCA generates hundreds ofrandomly-linked copies of a covalently closed circle in several minutes.If solid support matrix-associated, the DNA product remains bound at thesite of synthesis, where it may be labeled, condensed, and imaged as apoint light source. For example, linear oligonucleotide probes, whichcan generate RCA signals, have been bound covalently onto a glasssurface. The color of the signal generated by these probes indicates theallele status of the target, depending upon the outcome of specific,target-directed ligation events. As RCA permits millions of individualprobe molecules to be counted and sorted, it is particularly amenablefor the analysis of rare somatic mutations. RCA also shows promise forthe detection of padlock probes bound to single-copy genes incytological preparations.

In addition, a solid-phase RCA methodology has also been developed toprovide an effective method of detecting constituents within a solution.Initially, a recognition step is used to generate a complex h a circulartemplate is bound to a surface. A polymerase enzyme is then used toamplify the bound complex. RCA uses small DNA probes that are amplifiedto provide an intense signal using detection methods, including themethods described in more detail below.

-   Other examples of isothermal amplification systems include,    e.g., (i) self-sustaining, sequence replication (see e.g., Guatelli,    et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), (ii) the Qβ    replicase system (see e.g., Lizardi, et al., 1988. BioTechnology 6:    1197-1202), and (iii) nucleic acid sequence-based amplification    (NASBA™; see Kievits, et al., 1991. J. Virol. Methods 35: 273-286).

Amplification of a nucleic acid template as described above results inmultiple copies of a template nucleic acid sequence covalently linked toan anchor primer. In one embodiment, a region of the sequence product isdetermined by annealing a sequencing primer to a region of the templatenucleic acid, and then contacting the sequencing primer with a DNApolymerase and a known nucleotide triphosphate, i.e., dATP, dCTP, dGTP,dTTP, or an analog of one of these nucleotides. The sequence can bedetermined by detecting a sequence reaction byproduct, as is describedbelow.

The sequence primer can be any length or base composition, as long as itis capable of specifically annealing to a region of the amplifiednucleic acid template. No particular structure for the sequencing primeris required so long as it is able to specifically prime a region on theamplified template nucleic acid. Preferably, the sequencing primer iscomplementary to a region of the template that is between the sequenceto be characterized and the sequence hybridizable to the anchor primer.The sequencing primer is extended with the DNA polymerase to form asequence product. The extension is performed in the presence of one ormore types of nucleotide triphosphates, and if desired, auxiliarybinding proteins.

The method comprises the steps of: (a) introducing the template nucleicacid polymer into a polymerization environment in which the nucleic acidpolymer will act as a template polymer for the synthesis of acomplementary nucleic acid polymer when nucleotides are added; (b)successively providing to the polymerization environment a series offeedstocks, each feedstock comprising a nucleotide selected from amongthe nucleotides from which the complementary nucleic acid polymer willbe formed, such that if the nucleotide in the feedstock is complementaryto the next nucleotide in the template polymer to be sequenced saidnucleotide will be incorporated into the complementary polymer andinorganic pyrophosphate will be released; (c) separately recovering eachof the feedstocks from the polymerization environment; and (d) measuringthe amount of inorganic pyrophosphate by utilizing an ATP generatingpolypeptide-ATP converting polypeptide fusion protein in each of therecovered feedstocks to determine the identity of each nucleotide in thecomplementary polymer and thus the sequence of the template polymer.

The sequence primer can be any length or base composition, as long as itis capable of specifically annealing to a region of the amplifiednucleic acid template. No particular structure is required for thesequencing primer so long as it is able to specifically prime a regionon the amplified template nucleic acid. Preferably, the sequencingprimer is complementary to a region of the template that is between thesequence to be characterized and the sequence hybridizable to the anchorprimer. The sequencing primer is extended with the DNA polymerase toform a sequence product. The extension is performed in the presence ofone or more types of nucleotide triphosphates, and if desired, auxiliarybinding proteins.

This invention also includes a method wherein the amount of inorganicpyrophosphate is measured by (a) adding adenosine-5′-phosphosulfate tothe feedstock; combining the recovered feedstock containingadenosine-5′-phosphosulfate with an ATP generating polypeptide-ATPconverting polypeptide fusion protein such that any inorganicpyrophosphate in the recovered feedstock and theadenosine-5′-phosphosulfate will first react to the form ATP and sulfateand then react with luciferin in the presence of oxygen such that theATP is consumed to produced AMP, inorganic pyrophosphate, carbon dioxideand light; and (b) measuring the amount of light produced. In apreferred embodiment, the template polymer and ATP generatingpolypeptide-ATP converting polypeptide fusion protein are immobilized ona solid support.

The invention also includes a method for determining the nucleic acidsequence in a template nucleic acid polymer, comprising: (a) introducingthe template nucleic acid polymer into a polymerization environment inwhich the nucleic acid polymer will act as a template polymer for thesynthesis of a complementary nucleic acid polymer when nucleotides areadded; (b) successively providing to the polymerization environment aseries of feedstocks, each feedstock comprising a nucleotide selectedfrom among the nucleotides from which the complementary nucleic acidpolymer will be formed, such that if the nucleotide in the feedstock iscomplementary to the next nucleotide in the template polymer to besequenced said nucleotide will be incorporated into the complementarypolymer and inorganic pyrophosphate will be released; (c) separatelyrecovering each of the feedstocks from the polymerization environment;and (d) measuring the amount of PPi with a thermostable sulfurylase anda luciferase in each of the recovered feedstocks to determine theidentity of each nucleotide in the complementary polymer and thus thesequence of the template polymer. In one embodiment, the thermostablesulfurylase and the luciferase are joined in a fusion protein. Inanother embodiment, the thermostable sulfurylase is joined to anaffinity tag.

The invention further provides a method for sequencing a nucleic acid,the method comprising: (a) providing one or more nucleic acid anchorprimers; (b) providing a plurality of single-stranded circular nucleicacid templates disposed within a plurality of cavities on a planarsurface, each cavity forming an analyte reaction chamber, wherein thereaction chambers have a center to center spacing of between 5 to 200;(c) annealing an effective amount of the nucleic acid anchor primer toat least one of the single-stranded circular templates to yield a primedanchor primer-circular template complex; (d) combining the primed anchorprimer-circular template complex with a polymerase to form an extendedanchor primer covalently linked to multiple copies of a nucleic acidcomplementary to the circular nucleic acid template; (e) annealing aneffective amount of a sequencing primer to one or more copies of saidcovalently linked complementary nucleic acid; (f) extending thesequencing primer with a polymerase and a predetermined nucleotidetriphosphate to yield a sequencing product and, if the predeterminednucleotide triphosphate is incorporated onto the 3′ end of saidsequencing primer, a sequencing reaction byproduct; and (g) identifyingthe sequencing reaction byproduct with the use of a thermostablesulfurylase and a luciferase, thereby determining the sequence of thenucleic acid. In one embodiment, the thermostable sulfurylase and theluciferase are joined in a fusion protein. In another embodiment, thethermostable sulfurylase is joined to an affinity tag.

Also included in the invention is a method for sequencing a nucleicacid, the method comprising: (a) providing at least one nucleic acidanchor primer; (b) providing a plurality of single-stranded circularnucleic acid templates in an array having at least 400,000 discretereaction sites; (c) annealing a first amount of the nucleic acid anchorprimer to at least one of the single-stranded circular templates toyield a primed anchor primer-circular template complex; (d) combiningthe primed anchor primer-circular template complex with a polymerase toform an extended anchor primer covalently linked to multiple copies of anucleic acid complementary to the circular nucleic acid template; (e)annealing a second amount of a sequencing primer to one or more copiesof the covalently linked complementary nucleic acid; (f) extending thesequencing primer with a polymerase and a predetermined nucleotidetriphosphate to yield a sequencing product and, when the predeterminednucleotide triphosphate is incorporated onto the 3′ end of thesequencing primer, to yield a sequencing reaction byproduct; and (g)identifying the sequencing reaction byproduct with the use of athermostable sulfurylase and a luciferase, thereby determining thesequence of the nucleic acid at each reaction site that contains anucleic acid template. In one embodiment, the thermostable sulfurylaseand the luciferase are joined in a fusion protein. In anotherembodiment, the thermostable sulfurylase is joined to an affinity tag.

The invention also includes a method of determining the base sequence ofa plurality of nucleotides on an array, the method comprising: (a)providing a plurality of sample DNAs, each disposed within a pluralityof cavities on a planar surface, each cavity forming an analyte reactionchamber, wherein the reaction chambers have a center to center spacingof between 5 to 200 μm, (b) adding an activated nucleotide5′-triphosphate precursor of one known nitrogenous base to a reactionmixture in each reaction chamber, each reaction mixture comprising atemplate-directed nucleotide polymerase and a single-strandedpolynucleotide template hybridized to a complementary oligonucleotideprimer strand at least one nucleotide residue shorter than the templatesto form at least one unpaired nucleotide residue in each template at the3′-end of the primer strand, under reaction conditions which allowincorporation of the activated nucleoside 5′-triphosphate precursor ontothe 3′-end of the primer strands, provided the nitrogenous base of theactivated nucleoside 5′-triphosphate precursor is complementary to thenitrogenous base of the unpaired nucleotide residue of the templates;(c) detecting whether or not the nucleoside 5′-triphosphate precursorwas incorporated into the primer strands through detection of asequencing byproduct with a thermostable sulfurylase and luciferase,thus indicating that the unpaired nucleotide residue of the template hasa nitrogenous base composition that is complementary to that of theincorporated nucleoside 5′-triphosphate precursor; and (d) sequentiallyrepeating steps (b) and (c), wherein each sequential repetition addsand, detects the incorporation of one type of activated nucleoside5′-triphosphate precursor of known nitrogenous base composition; and (e)determining the base sequence of the unpaired nucleotide residues of thetemplate in each reaction chamber from the sequence of incorporation ofsaid nucleoside precursors. In one embodiment, the thermostablesulfurylase and the luciferase are joined in a fusion protein. Inanother embodiment, the thermostable sulfurylase is joined to anaffinity tag.

The invention will be further illustrated in the following non-limitingexamples. There are several abbreviations which will be used in thefollowing examples: FUS stands for fusion gene, S stands forsulfurylase, L stands for luciferase, TL stands for thermostableluciferase, X stands for XhoI, H stands for HindIII, N stands for NotIand B stands for BamHI. For example, FUS-L/S X F means a primer for thefusion gene, luciferase-sulfurylase Xho Forward and so forth. Primers 1through 6 are for the L or TL to S fusions and primers 7 through 13 arefor the S to L or TL fusions.

EXAMPLES Example 1 Cloning Strategy for Obtaining the Bst SulfurylaseGene

Gene specific primers, which incorporated restriction site linkers, weredesigned based on the sequence for a putative ATP sulfurylase fromBacillus stearothermophilus in ERGO, a curated database of genomic DNAmade available on the World Wide Web by Integrated Genomics whichincluded the Bacillus stearothermophilus Genome Sequencing Project atthe University of Oklahoma (NSF Grant #EPS-9550478). The forward primerutilized was 5′-CCC TTC TGC AGC ATG AGC GTA AGC ATC CCG CAT GGC GGC ACATTG-3′ (SEQ ID NO: 7) and the reverse primer used was 5′-CCC GTA AGC TTTTAG CGC GCT GAC GGG GCG ACC GTT TCG CGT TCT TG-3′ (SEQ ID NO:8). Thereaction mix for PCR amplification contained 5.0 uL 10× polymerasebuffer (Clontech, Cat. #8714), 2.0 uL 5 M betaine (Sigma, Cat. #B0300),1.0 uL dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP), 0.8 uL Advantage 2polymerase (Clontech, Cat. #8714), 0.2 uL Advantage-HF 2 polymerase(Clontech, Cat. #K1914), 10 pmol forward primer, 10 pmol reverse primer,100 ng (or less) Bst genomic DNA (ATCC, Cat. #12980D), and enoughdistilled water to make total volume of 50 uL. As little as 1 ng Bstgenomic DNA was sufficient to yield PCR product. The PCR amplificationof Bst ATP sulfurylase gene from genomic DNA consisted of an initialstep at 96° C. for 3 min, then 35 cycles of 96° C. for 15 sec, 60° C.for 30 sec, 72° C. for 6 min, a finishing step at 72° C. for 10 min andfinally 14° C. until removal. The PCR product was cleaned using QIAquickPCR Purification Kit (QIAGEN).

Example 2 Cloning Strategy for Obtaining the Sulfurylase-luciferaseFusion Protein

All chemicals were purchased from Sigma unless noted otherwise.Racemically pure D-luciferin was ordered from Pierce. The assay bufferfor measuring ATP sulfurylase and luciferase activities contained Taqpolymerase. A polymerase chain reaction (PCR)-mediated approach wasutilized to link the open reading frames (ORFs) of luciferase andsulfurylase. The cloning strategy is outlined in FIG. 1. Briefly, itinvolved the amplification of luciferase and sulfurylase ORFs by PCR,using primers that contain convenient restriction sites (XhoI andHindIII) to clone the fusion gene into an expression vector, in-frameand, the design of a rare restriction site (NotI) at the junction of thetwo polypeptides so that other versions of luciferase, such asthermostable luciferase (TL), and sulfurylase can be convenientlyswapped to obtain either sulfurlyase-luciferase (S-L) orluciferase-sulfurlyase (L-S) fusion proteins. A NotI site was used tofuse the variable heavy chain of antibodies to luciferase to generate aviable fusion protein. These primers were also designed in such a waythat the primers that form part of the junction of the two ORFs containsufficient overlapping regions of nucleotides. For example, the 5′ endof FUS-L/S Not R contains deoxynucleotides in an anti-parallelorientation that encode the N-terminal 10 amino acids of yeastsulfurylase. Thus, a PCR product generated using this primer wouldanneal to the 5′ end of yeast sulfurylase ORF and would generate thefusion protein, L-S.

The products in boxes were obtained by PCR as elaborated in FIG. 2. Asshown in FIG. 3, the PCR products were subjected to electrophoresis. ThePCR products were then purified, digested with XhoI and Hind III andsubcloned into XhoI/HindIII digested pRSETA-BCCP. pRSETA-BCCP is aderivative of pRSET A (Invitrogen) in which the sequence between NheIand BamHI restriction sites has been replaced by the portion of thebiotin carboxyl carrier protein (BCCP) gene from E. coli (GenBankaccession #M80458) that codes for residues 87-165. The 87-amino acidBCCP domain was obtained by PCR and cloned into the NheI and BamHI sitesof pRSETA to obtain pRSETA-BCCP. The ligated fusion protein andpRSETA-BCCP were transformed into BL21DE3 and TOP10 cells. BL21DE3 cellsyielded colonies for L-S and TOP10 cells yielded colonies for TL-S.

The following list of primers was used to construct the fusion proteins:

SEQ PRIMER ID NO TITLE NUCLEIC ACID SEQUENCE NO 1 FUS-L/S X F CCCC CTCGAG ATC CAA ATG GAA  9 GAC GCC AAA AAC ATA AAG AAA GGC CC 2 FUS-TL/S XCCCC CTC GAG ATC CAA ATG GCT 10 F GAC AAA AAC ATC CTG TAT GGC CC 3FUS-L/S Not TTG TAG AAT ACC ACC GTG AGG 11 R AGC AGG CAT AGC GGC CGC CAATTT GGA CTT TCC GCC CTT CTT GGC C 4 FUS-TL/S TTG TAG AAT ACC ACC GTG AGG12 Not R AGC AGG CAT AGC GGC CGC ACC GTT GGT GTG TTT CTC GAA CAT C 5FUS-S-Not F GCG GCC GCT ATG CCT GCT CCT 13 CAC GGT GGT ATT CTA C 6FUS-S-Hind CCCC AAG CTT TTA AAA TAC AAA 14 III R AAA GCC ATT GTC TTC CAAGAA TAG GAC 7 FUS-S/L B F CCCC GGA TCC ATC CAA ATG CCT 15 GCT CCT CACGGT GGT ATT CTA CAA GAC 8 FUS-S/L R GGGCCTTTCTTTATGTTTTTGGCGTCTT 16 CCATAGC GGC CGC AAA TAC AAA AAA GCC ATT GTC 9 FUS-L-F GCG GCC GCT ATG GAAGAC GCC 17 AAA AAC ATA AAG AAA GGC CC 10 FUS-L-N-R CCCC CCA TGG TTA CAATTT GGA 18 CTT TCC GCC CTT CTT GGC C 11 FUS-S/TL R GG GCC ATA CAG GATGTT TTT 19 GTC AGC CAT AGC GGC CGC AAA TAC AAA AAA GCC ATT GTC 12FUS-TL-F GCG GCC GCT ATG GCT GAC AAA 20 AAC ATC CTG TAT GGC CC 13FUS-TL-H-R CCCC AAG CTT CTA ACC GTT GGT 21 GTG TTT CTC GAA CAT CTG ACG C

These primes were utilized to perform PCR. The following PCR conditionwas used.

PCR Condition

-   96° C. for 96° C. for 0:15; 76° C. for 0:30; −1° C. per cycle;    72° C. for 6:00;-   For 15 cycles; 96° C. for 0:15; 60° C. for 0:30; 72° C. for 6:00;-   For 29 cycles; 72° C. for 10:00;-   14° C. forever

Example 3 Cloning of the His6-BCCP Bst ATP Sulfurylase Fusion Protein

The Bst-affinity tagged fusion construct is a derivative of pRSETA inwhich the NheI-XhoI fragment has been replaced by the BCCP domain andthe ATP sulfurylase is inserted after the BCCP domain.

Briefly, the BstSulf PCR product, as described in Example 1, wasdouble-digested with PstI and HindIII, isolated on a 1% agarose/TAE gel,purified using QIAEXII (QIAGEN) and ligated into the large PstI/HindiIIIfragment of pRSETA-BCCP using the Quick Ligation Kit from NEB accordingto manufacturer's instructions. As mentioned in Example 2, pRSETA-BCCPis a derivative of pRSET A (Invitrogen) in which the sequence betweenNheI and BamHI restriction sites has been replaced by the portion of thebiotin carboxyl carrier protein (BCCP) gene from E. coli (GenBankaccession #M80458) that codes for residues 87-165. 2 μL ligationreaction was used to transform 50 uL TOP10 competent cells (Invitrogen)and plated on LB-Ap plates. Sequencing of plasmid insert from ten cloneswas used to determine the consensus sequence for the ATP sulfurylasegene from ATCC 12980.

The plasmid pRSETA-BCCP-BstSulf was transformed into the E. coliexpression host BL21 (DE3)pLysS (Novagen) and the induction expressionof BstHBSulf was carried out according to the maufacturer'sinstructions. The cells were harvested and stored as frozen pellets. Thepellets were lysed using BugBuster plus Benzonase according tomanufacturer's instructions and protein was purified on a 20 mL columnpacked with Chelating Sepharose Fast Flow (Amersham, Cat. #17-0575-02)and charged with nickel (II). Protein was eluted using a 0-500 mMimidazole gradient. Analysis by SDS-PAGE showed a single band of thecorrect size.

Example 4 Binding Enzymes to Beads

The BCCP domain enables the E. coli to add a single biotin molecule ontoa specific lysine residue. Hence these fusion proteins can be bound tosolid supports that contain streptavidin. TL-S was successfully clonedinto a TA vector. 25 μl of MPG-Streptavidin (CPG, Inc.) orNickel-agarose (Qiagen) were taken in a 1.5 ml tube and placed on amagnet. The supernatant was removed and the beads were resuspended in 25μg of His6-BCCP-sulfurylase and 75 μg of His6-BCCP-luciferase. To testthe fusion protein, 100 μl of dialyzed fusion protein was bound to the25 μl of beads. The beads were allowed to mix at room temperature for 1hr, washed with assay buffer (25 mM Tricine (pH 7.8), 5 mM MgAcetate, 1mM DTT, 1 mM EDTA, and 1 mg/ml BSA) and assayed for enzyme activitieswith 1 mM PPi, 4 mM APS and 300 mM D-luciferin. With the nickel-agarosebeads, the EDTA was omitted from the assay buffer.

As shown in FIG. 3, these fusion proteins displayed activity on both theNTA- Agarose and MPG-SA beads. S:L 1:3 represents sulfurylase andluciferase bound individually to beads in a 1:3 ratio. Ni-Ag and MPG-SAare nickel-agarose and MPG-Streptavidin beads, respectively. PL isPromega luciferase, which does not have a polyhistidine or a biotin tagon it and hence serves as a negative control. Fraction 19 contains thefusion protein and is active on both kinds of beads. This suggests thatthe fusion protein was synthesized with a poly-histidine tag and abiotin molecule on the BCCP domain of the fusion protein.

1. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:2, 4 and
 6. 2. The polypeptideof claim 1 wherein the polypeptide comprises SEQ ID NO:2.
 3. Thepolypeptide of claim 1 attached to a mobile support.
 4. The polypeptideof claim 3 wherein the polypeptide is attached to the mobile support bya covalent or non-covalent interaction.
 5. The polypeptide of claim 4wherein the polypeptide is attached by a linkage selected from the groupconsisting of a CO²+-hexahistidine linkage, a Ni²⁺-hexahistidine linkagea biotin binding protein/biotin linkage, a glutathioneS-transferase/glutathione linkage, a monoclonal antibody/antigenlinkage, a maltose binding protein/maltose linkage and pluronic linkage.6. The polypeptide of claim 5 wherein the biotin binding protein isselected from the group consisting of streptavidin and avidin.
 7. Thepolypeptide of claim 3 wherein the mobile support is selected from thegroup consisting of a bead, optical fiber and glass surface.
 8. Thepolypeptide of claim 7 wherein the bead is a nickel-agarose bead or amagnetic porous glass Streptavidin bead.